Inhibiting Firefly Bioluminescence by Chalcones

Article abstract of DOI:10.1021/acs.analchem.7b00813

Chalcone refers to an aromatic ketone and an enone that constitutes the central core for various important biological compounds in drug discovery. Moreover, the firefly luciferase (Fluc) as the bioluminescent reporter has been widely used in life science

Full text of DOI:10.1021/acs.analchem.7b00813

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)
Article
Inhibiting the firefly bioluminescence by chalcones
Huateng Zhang, Jing Su, Yuxin Lin, Haixiu Bai, Jiaxiang Liu, Hui Chen, Lupei Du, Lichuan Gu, and Minyong Li
Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 11 May 2017
Downloaded from http://pubs.acs.org on May 12, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth
Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 9
Analytical Chemistry
1
2
3
4
5
6
7
8
Inhibiting the firefly bioluminescence by chalcones
Huateng Zhang, a,‡ Jing Su, b,c,‡Yuxin Lin, a Haixiu Bai,a,d Jiaxiang Liu,a Hui Chen,a Lupei Du, a
Lichuan Gub,* Minyong Lia,*
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong
University, Jinan, Shandong 250012, China. Tel./fax: +86-531-8838-2076; E-mail: mli@sdu.edu.cn
b State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong
250100, China. Tel./fax: +86-531-8836-2039; E-mail: lcgu@sdu.edu.cn
c Faculty of Light Industry, Province Key Laboratory of Microbial Engineering, Qilu University of Technology, Jinan,
Shandong 250353, China
d Shandong Medicine Technician College, Taian, Shandong 271016, China
‡These authors contributed equally.
ABSTRACT: Chalcone refers to an aromatic ketone and an enone that constitutes the central core for various important
biological compounds in drug discovery. Moreover, the firefly luciferase (Fluc) as the bioluminescent reporter has been
widely used in life science research and high-throughput screening (HTS). However, Fluc might suffer from direct inhibi-
tion by HTS compounds resulting in the occurrence of “false positives.” In the current research, we discovered a series of
chalcone compounds as Fluc inhibitors with favorable potency both in vitro and in vivo. Moreover, our compound 3i
showed remarkable systemic inhibition in transgenic mice. Both enzymatic kinetics study and cocrystal structure demon-
strated that compound 3i is competitive for substrate aminoluciferin, while noncompetitive for ATP. Besides, compound
3i exhibited excellent selectivity as a promising quenching agent in a simulated dual-luciferase reporter assay. We be-
lieved that our research would contribute to improving scientists’ awareness of the Fluc inhibitors, pay attention to the
bias results and even expand the utilization of bioluminescence in life science research.
INTRODUCTION
pharmacological targets is a critical approach to drug dis-
covery. Artese et al. performed an in silico HTS from
commercially natural compounds and obtained a chal-
cone derivative that could significantly inhibit the te-
lomerase activity7. More importantly, fully automated
HTS systems based on luciferase enzyme as biolumines-
cent reporters have become widely utilized in chemical
biology and drug discovery applications8.
Chalcone is widely found in nature and existed in licorice,
saffron, and other medicinal plants. Chalcone is not only
the main component of natural products, but also a
necessary precursor for synthetic manipulations. The core
scaffold of chalcone is 1,3-diphenylpropenone (Fig. 1).
Since the backbone structure of chalcones has greater
flexibility and is capable of binding to different receptors,
chalcones exhibit a broad range of therapeutic activity,
such as anti-cancer, anti-parasitic, anti-HIV, antiviral,
anti-fungal, antimalarial, anti-inflammatory and so on1-3.
This fact has attracted the widespread concern of re-
searchers, and they have found some promising com-
pounds by acting at different targets. For example, chal-
cones can promote tumor cell apoptosis, inhibit angio-
genesis and cell proliferation4. Zhang et al. synthesized
xanthohumol (Xn), a natural product derived from hops,
and its analogs, then discovered that an analog showed
the highest cytotoxicity toward HeLa cells, with IC50 val-
ues of 1.4 μM5. Ram et al. synthesized oxygenated chal-
cone and bischalcone and exhibited excellent antimalarial
activity against Plasmodium berghei in mice6. It is note-
worthy that high-throughput screening (HTS), a relatively
recent innovation, is increasingly being applied to drug
discovery enterprise. HTS of compound libraries against
Figure 1. The structure of chalcone (A) and resveratrol
(B)
Bioluminescence is a natural phenomenon through an
enzyme-catalyzed reaction with the emission of visible
light in a living organism. The firefly luciferin–luciferase
system, the most widely applied bioluminescent system,
has been well studied for more than 50 years. D-Luciferin
(LH2) can be oxidized into oxyluciferin under the partici-
pation of Fluc with an emission wavelength from 550 to
620 nm. Bioluminescence assay can be carried out in ab-
ACS Paragon Plus Environment

Analytical Chemistry
solute darkness without the demand of excitation light
Page 2 of 9
inhibition to obtain Km and Vmax values after incubation
with different concentrations of compound 3i. In brief,
compound 3i was competitive for aminoluciferin while
noncompetitive for ATP. These results were consistent
with the subsequent cocrystal structure of luciferase
complex with compound 3i. The cocrystal result also
demonstrated that compound 3i was bound in a pocket
1
2
3
4
5
6
7
8
sources; thus, it can avoid the effect of the emission light
and has a higher sensitivity compared with fluorescence
methods9. FLuc-based bioluminescence assays are highly
favored in HTS because of their excellent sensitivity10.
However, the luciferase enzymes commonly used in the
HTS may suffer from unexpected nonspecific activation or
inhibition results. Consequently, it is essential to investi-
gate the molecular mechanisms to achieve high accuracy
and reliability11.
consisting of luciferase residues and occupied the catalyt-
ic active site of amino-luciferin. More interestingly, com-
pound 3i could selectivity quench Fluc bioluminescence,
which could be utilized as a promising quenching agent
in a dual-luciferase reporter assay.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In 1965, Ueda et al. firstly reported that general
anesthetics, such as diethyl ether and halothane,
inhibited the bioluminescence of firefly luciferase.12 Fraga
et al. found that coenzyme A induced stabilization of Fluc
luminescence, because a thiolytic reaction splited
dehydroluciferyl-adenylate(L-AMP) and generated
dehydro-luciferyl-CoA(L-CoA), a much less powerful
inhibitor13. In 2006, Bakhtiarova et al. suggested that
resveratrol (Fig. 1B) could inhibit the firefly luciferase
activity significantly with a Ki value of 2 μM, proposing
that resveratrol might exist fundamentally flawed via
luciferase reporter assays.14 Auld et al. reported PTC124 (3-
[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) as a
potent Fluc inhibitor with an IC50 of 7 nM15,16. Rui Fontes
et al. indicated that the bioluminescence of luciferin
could be inhibited by inorganic pyrophosphate and
tripolyphosphate due to their reaction with L-AMP 17. So
far, many compounds containing different structures have
been reported that could inhibit firefly luciferase, such as
ions,18 (E)-2-fluoro-4’-methoxystilbene19, N-pyridin-2-
ylbenzamides20, 2-phenylnaphthalenes21, aryltriazoles22, 2-
phenylnaphthalenes23, 5-benzyl-3-phenyl-4,5-
EXPERIMENTAL SECTION
Synthesis
Scheme 1. Synthesis of a series of chalcones by aldol con-
densation reaction. Reagents and conditions: (c) TsOH,
AcOH, 85 °C, 12 h, 33-80%; (d) H2SO4, CH3OH, 65 °C, 6 h,
88%; (e) NaBH4, Pd/C, THF, H2, rt, 4 h, 60%.
The synthetic route of chalcone compounds is outlined
in Scheme 1. In brief, we started from the commercially
available different substituted acetophenone or made
some simple modification according to the method de-
scribed by and Huang and Ley 24,25. Then, the chalcone
compounds were obtained by toluene sulfonic acid-
catalyzed or sodium hydroxide-catalyzed aldol condensa-
tion reaction. In order to delve into their structure-
activity relationship, we further performed the esterifica-
tion of carboxylic acids and the reduction of α,β-
unsaturated ketones. More synthetic details can be found
in the supporting information.
dihydroisoxazoles and 5-benzyl-3-phenyl-1,4,2-
dioxazoles11.
In the current study, we found that some chalcone de-
rivatives could potently inhibit the firefly luciferase activi-
ty. Accordingly, it is of great significance to notify re-
searchers of possible “false positives” when they use FLuc-
based HTS to do some research on chalcones. We used
resveratrol as the positive control to evaluate the activity
of our chalcone compounds. In the current case, ami-
noluciferin was employed to study the kinetics profile of
inhibition of firefly luciferase due to its higher affinity
than the nature luciferin and longer emission wavelength
(596 nm), which is beneficial to in vivo inhibition study9.
We found (E)-5-[3-oxo-3-(4-bromophenyl)-1-propenyl]-2-
hydroxy-benzoic acid (compound 3i) inhibited firefly
luciferase with an IC50 value of 0.2 μM in vitro and (E)-5-
[3-oxo-3-(4-phenylphenyl)-1-propenyl]-2-hydroxy-benzoic
acid(compound 3f) inhibited firefly luciferase with an IC50
value of 2.49 μM in cellulo, which significantly stronger
than resveratrol (2.33 μM in vitro and 35.94 μM in
Clone, protein expression and purification of firefly
luciferase
Luciferase gene was amplified by PCR using Pgex-6p-2
plasmid contains the cDNA of Photinus pyralis luciferase.
The synthetic primers used as follows (the restriction sites
are underlined): forward primer, ATCggatccATGGAG-
GATGCGAAG, reverse primer, TCA ctcgagTTACAGTTT-
GCTTTTACC. The PCR product was cloned into the
BamHI, and XhoI sites of pET-15b (Novagen) and the
plasmid was overexpressed in E. coli strain BL21 (DE3).
The E.coli cells were cultured in the LB medium contain-
ing 100 μg/mL ampicillin at 310 K to an absorbance (A600)
reached 1.0 and were then induced with 1 mM IPTG (iso-
propyl β-D-1-thiogalactopyranoside) overnight at 288 K.
The cells were harvested by centrifugation. Cells lysis was
achieved by sonication method. After centrifugation at
28,000 × g for 45 min, the supernatant was applied to Ni-
cellulo).We further evaluated the inhibitory activity of
compound 3i in vivo and found that it exhibited 60.0%
inhibition in xenografted balb/c-nu female mice by an
intratumor injection and 18.8% inhibition in CAG-luc-
eGFPL2G85 transgenic mice by a tail intravenous injec-
tion. Moreover, we investigated its kinetic mechanism of
ACS Paragon Plus Environment

Page 3 of 9
Analytical Chemistry
serum (FBS) at 37°C in a humidified atmosphere with 5%
NTA column (GE Healthcare) for affinity chromatog-
1
2
3
4
5
6
7
8
raphy. The His-tagged luciferase was eluted with elution
buffer (10 mM Tris–HCl pH 8.0, 500 mM NaCl, and
250mM imidazole). The purification process was then
followed by size-exclusion chromatography with Super-
dex-200 column with 10 Mm Tris-HCl buffer pH 8.0 con-
taining 500 mM NaCl and 0.3 mM dithiothreitol.
CO2 incubator. In ES-2-FLuc cell bioluminescence inhibi-
tion assays, 100 μL cell suspension were added to a 96-
well plate (Corning, 3603) to make the number of cells in
each well at about 4×104. After incubated for 12 hours, 100
μL chalcone compounds of different concentrations (15.6
μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500μM, 1 mM)
dissolved by RPMI 1640 medium without FBS were added
as triplicates. Moreover, an equal amount of RPMI 1640
medium without compounds was added directly as a neg-
ative control. After further incubation for 12 hours, the
medium was removed, and 100 μL amino-luciferin solu-
tion (100 μM, dissolved in Tris buffer of pH 7.60) was
added. Also, the blank group (without cells) containing
only 100 μL aminoluciferin solution were set. After the
aminoluciferin solution had been added, the biolumines-
cence signal was measured immediately with IVIS Kinetic
(Caliper Life Sciences, USA) instrument equipped with a
cooled charge-coupled device (CCD) camera for biolumi-
nescent imaging. The exposure time was set to one se-
cond. Circular regions of interest(ROI) were drawn and
quantified using Living Image software.The data were
reported as total photon flux within an ROI in photons
per second and were analyzed using the GraphPad Prism
software.
Bioluminescence assay materials and instruments
9
Ultrapure water purified in a Mill-Q filtration system was
used to prepare all aqueous solutions. Measurements for
bioluminescent assays were performed in 50 mM Tris
buffer, pH 7.46 with 10 mM MgSO4 at 37°C. Recombinant
Renilla reniformis luciferase was purchased from RayBi-
otech. ATP and Tris base were purchased from Aladdin.
Coelenterazine was purchased from Chemedir. Lumines-
cence produced by the luciferase was measured with
Omega microplate reader (POLARstar Omega, Germany).
Bioluminescence imaging was performed using an IVIS
Kinetic (Caliper Life Sciences, USA) equipped with a
cooled charge-coupled device (CCD) camera. Circular
regions of interest (ROI) were drawn and measured using
Living Image software. The intensity of luminescence was
reported as total photon flux within an ROI in photons
per second.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Mice model
Luciferase enzyme inhibition assay
Balb/c-nu female mice were purchased from Animal Cen-
ter of China Academy of Medical Sciences when they were
8 weeks of age. To generate tumor xenografts in nude
mice, ES-2-Fluc cells (1×107) were implanted subcutane-
ously under the right armpit region. Mice were kept in a
standard 12:12 light–dark cycle either single- or group-
housed with free access to food and water at 28 °C. Tu-
mors were allowed to grow for two weeks before imaging.
Transgenic mice harboring the CAG-luc-eGFP L2G85
transgene, constructed by the laboratory of Dr. Christo-
pher H. Contag (Stanford University School of Medicine),
were purchased from the Jackson Laboratory (Sacramen-
to, California, USA). All animal studies were approved by
the Ethics Committee of Qilu Health Science Center,
Shandong University and complied with European guide-
lines for the care and use of laboratory animals.
Measurements for bioluminescent assays were performed
in 50 mM Tris buffer, pH 7.60 with 10 mM MgSO4 at 37
°C. All chalcone compounds were dissolved in dimethyl-
sulfoxide at 50 mM, then they were further diluted to an
increasing concentration ranging from 10 nM to 100 μM in
Tris buffer prepared before. The recombinant firefly lucif-
erase was purchased from Promega (E1702, USA). The
luciferase was diluted to 20 μg/mL inTris buffer. Sub-
strates solution is also prepared in the Tris buffer, con-
taining 40 μM of aminoluciferin and 2 mM of ATP. To a
96-well plate (WHB, black) each well containing 50 μL of
the luciferase solution, an amount of 50 μL of increasing
concentrations of chalcone compounds was added as
three replicates. After incubation at 25°C for 10 min, an
amount of 100 μL of the substrates solution was added.
An equal amount of DMSO in Tris buffer instead of com-
pounds was set as a control group. Instead of compounds
and luciferase, 100 μL Tris buffer was set as a blank group.
Luminescence produced by the luciferase was measured
with Omega microplate reader (POLARstar Omega, Ger-
many). The log-(inhibitor) versus normalized response
data were analyzed with the GraphPad Prism soft-
ware(control was set as 100%).
Bioluminescence inhibition assay in mouse xeno-
graft model
Day 1: Fifteen mice bearing ES-2-Fluc subcutaneous tu-
mors were randomly divided into 3 groups (n=5, each):
normal saline group, resveratrol group, inhibitor 3i group.
The mice were anesthetized with isoflurane and then in-
traperitoneally injected with 100 μL aminoluciferin (0.5
mM, diluted by normal saline), followed immediately by
bioluminescence imaging every 3 minutes for 30 to 40
minutes until the bioluminescence intensity reached a
peak and got steady. Then, mice were fed with regular
diet for 12 hours to metabolize away the aminoluciferin.
After that, for inhibitor 3i group and resveratrol group,
each mouse was injected with 50 μL resveratrol or 3i (200
μM) intratumorally, and for the normal saline group, 50
ES-2-Fluc cell bioluminescence inhibition assay
ES-2 cells (human ovarian cancers cell line) were
purchased from the Committee on Type Culture Collec-
tion of Chinese Academy of Sciences. ES-2 cells
expressing firefly luciferase (ES-2-Fluc cells) were
supplied by Cellcyto. The ES-2-Fluc cells were cultured in
RPMI 1640 medium supplemented with 10% fetal bovine
ACS Paragon Plus Environment

Analytical Chemistry
Page 4 of 9
μL normal saline containing an equal amount of DMSO min. The obtained crystal was flash-frozen in liquid nitro-
1
2
3
4
5
6
7
8
was used to instead of inhibitors.
gen after soaking in a cryoprotectant solution consisting
of the respective reservoir solution with 15-20% glycerol
was used as a cryoprotectant, and all data were collected
at 100 K in a nitrogen stream.
Day 2: After feeding for another 12 hours (on the next
day), all mice were intraperitoneally injected with 100 μL
aminoluciferin (0.5mM, diluted by normal saline). Subse-
quently, bioluminescent imaging was performed every 3
minutes for 30 to 40 minutes. The relative activity for
each mouse was calculated by dividing the peak total
photon flux of day 1 by peak total flux of day 2. The resid-
ual activity of mice treated with inhibitors is calculated by
the ratio of comparing relative activity of inhibition group
with the saline group, setting the saline group as 100%.
Data collection, processing and structure determina-
tion
X-ray diffraction data were collected at 100 K on beamline
BL17U at SSRF, Shanghai, China equipped with a MAR
Mosaic CCD 225 detector. The data were integrated and
scaled using the HKL-200 program suite.26 The crystals of
Fluc-3i belong to space group P41212 with unit cell param-
eters: a=118.141 Å, b=118.141 Å, c=96.194 Å, α=β=γ=90° dif-
fracted to 3.0 Å resolution. The structure of Fluc-3i was
solved by molecular replacement using Phaser from the
CCP4 suite of program 27,28 with firefly luciferase (PDB
entry 1BA3) as the search model. The initial model of lu-
ciferase was refined using PHENIX29 with additional
rounds of manual rebuilding using the Coot molecular
graphics program.30 The compound 3i was added to the
complex model by Coot based on the FO-FC density map
of the ligand structure. The final R values were
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In vivo inhibition assay in transgenic mice by tail
intravenous injection
Pathogen-free luciferase-expressing transgenic mice
(FVB-Tg(CAG-luc,-GFP)L2G85Chco/FathJ)17 were ob-
tained from the Jackson Laboratory and housed in the
Shandong University. Twelve transgenic mice were
randomized into three groups: compound 3i group,
resveratrol group, and normal saline group. For inhibition
groups, 200 μL compound 3i or resveratrol (200 μM, dis-
solved in normal saline) were injected into the mice veins
through tail intravenous injection. For the normal saline
group, we used 200 μL normal saline instead. After 4
hours, the mice were anesthetized with isoflurane and
injected with 100 μL amino-luciferin (0.5 mM, dissolved in
normal saline) intraperitoneally. After injection with ami-
no-luciferin, bioluminescent imaging was taken immedi-
ately every 3 minutes for 30 to 40 minutes until the bio-
luminescence intensity went through a peak and got
steady. The inhibition rate was obtained by comparing
the average bioluminescence flux of inhibition group with
the normal saline group.
Rwork=0.1954 and Rfree=0.2698 based on a subset of 14148 of
the reflections.
X-ray diffraction data collection and refinement statis-
tics are presented in Table S1. The final model was
checked and validated using PROCHECK31, QMEAN32 and
ProQ33, which indicated a good-quality model. The mean
temperature factors for protein and solvent were calculat-
ed using BAVERAGE from the CCP4 program suite. Mo-
lecular graphics was illustrated with PyMOL.34 The atomic
coordinates and structure factors of Fluc-3i have been
deposited in the Protein Data Bank with accession codes
5WYS.
Dual-luciferase reporter gene assay simulation
Statistical analysis
Firstly, Fluc and Rluc dissolved in Tris-HCl buffer (20
μg/mL and 2.5 μg/mL, respectively) was mixed in a 96-
well plate. Secondly, 50 μL Fluc substrates solution (20
μM aminoluciferin and 1 mM ATP in Tris-HCl buffer)
were added to the mixture to initiate the firefly lumines-
cence. Lastly, 100 μL mixture of compound 3i and coelen-
terazine (50 μM and 5 μM in Tris-HCl buffer, respectively)
was added to quench the firefly luminescence and trigger
renilla luminescence. Bioluminescence was measured
immediately at wavelength 590 nm and 460 nm, emitted
by Fluc and Rluc, respectively.
Data values were expressed as means ± SD or SEM of at
least two independent experiments and evaluated using
Student’s t-test for unpaired samples.
RESULTS AND DISCUSSION
In an initial screening, all chalcones were evaluated for
their inhibitory activity on purified recombinant firefly
(Photinus pyralis) luciferase. To determine their IC50
values and to generate the accurate concentration–
response curves (CRCs) (Fig. 2A), we used the increasing
concentrations of chalcones from 10 nM to 100 μM
incubation with firefly luciferase for 10 min and measured
the luminescence intensity with Omega microplate reader
(POLARstar Omega, Germany) while resveratrol served as
a positive control. As shown in Table S1, some chalcone
compounds showed significantly enhanced inhibitory
activity compared to the positive control resveratrol.
Among them, compound 3i showed the most potent in-
hibitory activity with an IC50 value of 0.2 μM and exceed-
ed 10-fold potency than the positive control resveratrol
(IC50=2.33 μM). Compared with other common inhibitors
in the assay, the IC50 value of compound 3i also exceeded
Crystallization of Fluc and Fluc-3i complex
Details of clone, expression and purification are described
in the Supporting Information. Luciferase was
concentrated to 8 mg/mL. Crystal of native luciferase was
initially obtained by sitting-drop vapor diffusion at 293 K.
After optimization, crystal was grown in hanging drops by
mixing equal volumes of protein solution and reservoir
solution (0.5 M Li2SO4, 15% PEG8000, 0.1 M Tris pH 8.0)
at 293 K. To obtain crystal of Fluc-3i complex, native
crystal was soaked in solution containing 1.2 mM
inhibitor, 3.2 mM ATP and 12 mM MgCl2 at 293 K for 1
ACS Paragon Plus Environment

Page 5 of 9
Analytical Chemistry
stituted group. Among them, bromine-substituted and
L-luciferin (IC50=115 μM), dehydroluciferin (IC50=2.3 μM),
1
2
3
4
5
6
7
8
and dehydroluciferyl coenzyme A (IC50=5 μM), but was no
better than L-AMP (IC50=6 nM) and so on35-37. Because the
luciferase-based assay was based on a readout of
luminescence intensity at 590 nm, the inhibition
quenching could cause the false-positive result. There-
fore, we determined the absorbance spectra (250-700 nm)
of compound 3i and FLuc emission spectrum in absence
or presence of compound 3i. As shown in Fig. 3B, com-
pound 3i could significantly decrease the luminescence
intensity while having no impact on the maximum emis-
sion wavelength of Fluc. Moreover, compound 3i did not
show any absorption at above 460 nm (Fig. 3). Therefore,
compound 3i was unable to absorb the visible light pro-
duced by Fluc.
iodine-substituted chalcones have a higher potency than
fluorine-substituted and chloro-substituted. This phe-
nomenon might be explained by the size of the atomic
radius.
Since the bioluminescence-based assay is widely applied
in vivo, we further evaluated inhibitory activity of com-
pound 3i in a well-established nude mice xenograft tumor
model described previously in detail by us21,38. To
9
construct the expression of firefly luciferase in nude mice
xenografts, ES-2-Fluc cells were grafted subcutaneously
into 8-week-old female mice under the right armpit re-
gion. After two weeks of tumor growth, bioluminescence
signal of total flux was measured after intraperitoneal
injection of amino-luciferin solution. The biolumines-
cence signal of the first day was set as the calibration val-
ue. After 12 h, we injected inhibitors (resveratrol and
compound 3i, 200 μM) into the tumor and measured its
bioluminescence intensity after waiting for another 12 h.
The relative activity was obtained by calculating the ratio
of the first and second day of bioluminescence intensity.
As shown in Fig. 4A, the ratio of the blank normal saline
group was increased to 135.3%, which may be due to tu-
mor growth for 24 h. Therefore, we set the saline group as
100% and obtained residual total flux percentage by calcu-
lating the ratio of the relative activity of inhibition group
(resveratrol and compound 3i) and the blank normal sa-
line group. As shown in Fig. 4B, the residual activity of
the bioluminescence was 40.0% after injection of com-
pound 3i, while the residual activity of the positive con-
trol resveratrol group was 66.5%. Inhibition rate of inhibi-
tion group can be calculated by 100% -residual total flux
(%). This indicated that compound 3i showed stronger
inhibitory activity than resveratrol, and our compound 3i
(200 μM) showed the inhibitory activity of 60.0%in the
nude mouse xenograft tumor model.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. (A) Concentration–response curves for active
chalcone compounds in a recombinant firefly luciferase
inhibition assay; (B) CRCs for active chalcone compounds
in cellulo. Representative graphs are selected from one
experiment performed in triplicate.
Figure 3. (A)Light absorbance spectrum for compound 3i
and (B) bioluminescence emission spectrum with or
without compound 3i
To investigate the effect of chalcones on firefly lucifer-
ase in cellulo, we performed a cell-based assay with ES-2-
Fluc ovarian cancer cells that transfected with firefly lu-
ciferase. ES-2-Fluc cells were treated with various in-
creased concentrations of inhibitors incubation for 12 h.
After incubated for 12 h, the medium was removed, and
the aminoluciferin solution was added. The correspond-
ing CRCs are shown in Fig. 2B. As shown in Table S1,
compound 3f exhibited potent inhibitory activity with an
IC50 value of 2.49 μM, that is >10-fold stronger than
resveratrol (IC50 =35.94 μM). Analysis of structure–activity
relationship (SAR) reveals that the exposed carboxyl
group on the right benzene ring is essential for maintain-
ing high potency. The esterification of carboxylic acids
will lead to the loss of activity. The activity of the carboxyl
group at meta-position is a little better than the para-
position. Hydroxy group at the R2 position is not essential
for the activity. Notably, the α, β-unsaturated ketone is
also necessary for inhibitory activity. Besides, substituted
group at the R1 position can increase the efficacy of inhibi-
tion, in which halogen is more favorable than other sub-
Figure 4. (A) Representative bioluminescence imaging in
a nude mice xenograft tumor model. Relative activity was
obtained by the total flux ratio of day 2 to day 1; (B) Quan-
tification of residual activity, calculated by the ratio of
relative activity of inhibition group to saline group. **
<0.01, * <0.05 (t-test, determined by GraphPad Prism
software).
ACS Paragon Plus Environment

Analytical Chemistry
significant decrease in Vmax, while the Km slightly de-
Page 6 of 9
1
2
3
4
creased. We supposed that the inhibition mode belonged
to noncompetitive inhibition. It revealed that compound
3i might combine with the luciferase outside the active
site.
5
6
7
8
Structures of luciferase complexed with compound 3i
were determined at 3.0 Å resolution by molecular re-
placement (see supporting information, Table S3) using
the crystal structure of luciferase from Photinus pyralis
(Protein Data Bank accession number: 1BA3) as a model.
The final models of Fluc-compound 3i show that each
asymmetric unit contains one monomer. All atoms of the
Fluc-compound 3i model are well defined except for two
amino-acid residues at the N-terminus, a loop from
Ser198 to Ser201 and seven residues at C-terminus.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Representative bioluminescence imaging of
inhibition caused by compound 3i (A) and resveratrol (B)
compared with normal saline in transgenic mice; (C)
quantification of inhibition rate caused by compound 3i
and resveratrol. ** <0.01 (t-test, obtained by GraphPad
Prism software).
To investigate whether our compound 3i can exhibit
systemic inhibition effects in the body, we further evalu-
ated its inhibition activity in CAG-luc-eGFP L2G85 trans-
genic mice by tail intravenous injection. The transgenic
mice directed by the CAG promoter can show wide-
spread expression of firefly luciferase in the whole body.
For the inhibition group, the transgenic mice were tail
vein injection of compound (200 μL of 200 μM solution in
saline); for the saline group, transgenic mice received 200
μL of saline instead. After 4 h, the bioluminescence inten-
sity was measured after intraperitoneal injection of ami-
noluciferin (100 μL, 0.5 mM, dissolved in saline). Inhibi-
tion rates of inhibitors can be calculated by 100% -
residual total flux (%). As depicted in Fig. 5, compound 3i
exhibited 18.8% inhibition in transgenic mice while the
positive control resveratrol exhibited only 10.1% inhibi-
tion. This phenomenon also demonstrated that com-
pound 3i showed more powerful inhibition than resvera-
trol in vivo.
Figure 6. The overall structure of Fluc-compound 3i
complex. (A): The whole Fluc-compound 3i complex
structure is represented in wheat color cartoon model;
compound 3i is drawn in green stick model and FO-FC
OMIT map contoured at 1.5 σ shows electron density for
compound 3i; (B): A schematic drawing of compound 3i
binding site with Ligplot program; (C): The interaction of
compound 3i with luciferase residues at the bottom of the
pocket; (D): The located sites comparison of Fluc-
compound 3i and Fluc-PTC124-AMP.
Apo-form crystals were soaked in the presence of com-
pound 3i and the co-crystal structure electron density
maps were examined for ligand binding (Fig. 6A). The
compound 3i is bound in a pocket consisting of luciferase
residues (Fig. 6B). Tyr255 and Asn229 forms hydrogen-
bonded with 3i at the bottom of the pocket (Fig. 6C). The
alignment of the Fluc-compound 3i structure to Fluc-
PTC124-AMP structure (Protein Data Bank accession
number: 3IES) indicated that the inhibitor 3i has the simi-
lar binding pattern with PTC-124. The hydrophobic pock-
et of Fluc is similar to PTC-124, except for a loop (Ala313-
Leu319) of the luciferase has about 3.5 Å distance move-
ment (Fig. 6D). These crystallographic results confirmed
the competitive pattern of compound 3i for aminolucifer-
in by kinetics study. The atomic coordinates and struc-
ture factors of Fluc-3i have been deposited in the Protein
Data Bank with accession codes 5WYS.
To determine the inhibition mode, we continued to
choose compound 3i for further concentration-dependent
response in the presence of aminoluciferin and ATP. Max-
imum rate and Michaelis constant with or without com-
pound 3i were determined with Michaelis−Menten
kinetics, and the Lineweaver–Burk plots were estimated
using GraphPad Prism software. For substrate aminolucif-
erin, the Michaelis constants (Km) were significantly
increased in the presence of the inhibitor, but the maxi-
mum rates (Vmax) remained unaltered (Fig. S1 and Table
S2). It indicated the inhibition mechanism for compound
3i was competitive with respect to the aminoluciferin.
Therefore, we believe that compound 3i can be bound to
the enzyme's active site where the substrate aminolucifer-
in also binds. For substrate ATP, compound 3i caused a
ACS Paragon Plus Environment

Page 7 of 9
Analytical Chemistry
3i inhibited firefly luciferase with an IC50 value of 0.2 μM
1
2
3
4
5
6
7
8
in vitro, approximately ten times greater potency than
resveratrol. Compound 3f inhibited firefly luciferase with
an IC50 value of 2.49 μM in cellulo, which also significant-
ly stronger than resveratrol. In the animal experiment,
compound 3i disclosed 60.0% inhibition in a well-
established nude mouse xenograft tumor model and
18.8% inhibition in CAG-luc-eGFP L2G85 transgenic mice.
In addition, kinetics of inhibition of luciferase by com-
pound 3i and the cocrystal structure also indicated that
compound 3i occupied the catalytic active site of sub-
strate aminoluciferin. In other words, compound 3i was
competitive for aminoluciferin while noncompetitive for
ATP. Furthermore, compound 3i achieved its inhibition in
an extremely rapid way when exposed to Fluc and had
little effect on Rluc, which might be used as a promising
quenching agent in a dual-luciferase reporter assay. Thus,
our studies can be helpful for research about chalcones in
drug discovery and might contribute to expanding its
application in life science research.
9
10
11
12
13
14
15
Figure 7. Dual-luciferase reporter assay simulation. Re-
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
sidual activity was obtained by RLU ratio of residual Fluc
to Fluc. ** <0.01 (t-test, calculated by GraphPad Prism
software).
The luciferase from the sea pansy Renilla reniformis
(Rluc), another commonly used luciferase reporter, has
been found some important applications in bioanalysis.39
It can catalyze the coelenterazine oxidation to coelen-
teramide with the emission of blue light.40 Rluc is com-
monly used together with Fluc in dual-luciferase reporter
gene assay. Both reporters can be examined from the
same sample by first measuring the Fluc activity and then
quenching Fluc luminescence and measuring the RLuc
activity following adding a quench-and-activate reagent.8
In the process of enzymatic inhibition assays, we found
that our chalcones inhibited firefly luciferase in an ex-
tremely rapid way. Therefore, we performed a dual-
luciferase reporter assay simulation to evaluate the lucif-
erase selectivity of compound 3i. First of all, Fluc sub-
strate solution containing aminoluciferin (20 μM) and
ATP (1 mM) were added into Fluc and Rluc mixture to
generate the firefly bioluminescence. Then, a mixture of
compound 3i (50 μM) and Rluc substrate coelenterazine
(5 μM) was added to the same sample to quench firefly
luminescence and trigger renilla luminescence. Biolumi-
nescence generated by the luciferase-catalyzed reactions
was measured immediately at wavelength 590 and 460
nm with an Omega microplate reader. As shown in Fig. 7,
for the positive control, we found resveratrol quenched
about 96% Fluc bioluminescence; however, it also elimi-
nated 33.4% Rluc bioluminescence. Compound 3i elimi-
nated nearly 98% of Fluc bioluminescence while having
less effect on Rluc bioluminescence compared to the
resveratrol. Therefore, compound 3i exhibited selectivity
which quenched Fluc bioluminescence while having little
impact on Rluc bioluminescence better than resveratrol.
It indicated that compound 3i had a high potential of be-
ing applied in the dual-reporter assay as a promising Fluc
quenching agent.
ASSOCIATED CONTENT
Supporting Information
General methods, synthetic procedures, original spectra for
structural characterization and additional figures and tables.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
(ML) Tel./fax: +86-531-8838-2076; E-mail: mli@sdu.edu.cn
(LG) Tel./fax: +86-531-8836-2039; E-mail: lcgu@sdu.edu.cn
Author Contributions
The manuscript was written through contributions of all
authors. ‡These authors contributed equally.
ACKNOWLEDGMENT
The present work was supported by grants from the
National Program on Key Basic Research Project of China
(Nos. 2013CB734000 and 2015CB150600), the National
Natural Science Foundation of China (Nos. 81673393 and
31401626), the Taishan Scholar Program at Shandong
Province, the Qilu Scholar Program at Shandong
University, the Program for Changjiang Scholars and
Innovative Research Team in University (No. IRT13028),
the Major Project of Science and Technology of Shandong
Province (No. 2015ZDJS04001), the Fundamental Research
Funds of Shandong University (No. 2014JC008) and the
National Institutes of Health (No. R01GM112003).
REFERENCES
(1) Dias, T. A.; Duarte, C. L.; Lima, C. F.; Proenca, M. F.;
Pereira-Wilson, C. Eur. J. Med. Chem. 2013, 65, 500-510.
(2) Ugwu, D. I.; Ezema, B. E.; Eze, F. U.; Onoabedje, E. A.;
Ezema, C. G.; Ekoh, O. C.; Ayogu, J. I. Int. J. ChemTech
Res. 2015, 7, 1966-1984.
CONCLUSION
In summary, we discovered a series of chalcone com-
pounds as potent firefly luciferase inhibitors. Compound
ACS Paragon Plus Environment

Analytical Chemistry
Page 8 of 9
(3) Gomez-Rivera, A.; Aguilar-Mariscal, H.; Romero-
(28) Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson,
E. J.; Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E.
B.; Leslie, A. G.; McCoy, A.; McNicholas, S. J.; Murshudov,
G. N.; Pannu, N. S.; Potterton, E. A.; Powell, H. R.; Read,
R. J.; Vagin, A.; Wilson, K. S. Acta. Crystallogr. D. Biol.
Crystallogr. 2011, 67, 235-242.
(29) Adams, P. D.; Grosse-Kunstleve, R. W.; Hung, L. W.;
Ioerger, T. R.; McCoy, A. J.; Moriarty, N. W.; Read, R. J.;
Sacchettini, J. C.; Sauter, N. K.; Terwilliger, T. C. Acta.
Crystallogr. D. Biol. Crystallogr. 2002, 58, 1948-1954.
(30) Emsley, P.; Cowtan, K. Acta. Crystallogr. D. Biol.
Crystallogr. 2004, 60, 2126-2132.
(31) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.;
Thornton, J. M. J. Appl. Crystallogr. 1993, 26, 283-291.
(32) Benkert, P.; Tosatto, S. C.; Schomburg, D. Proteins
2008, 71, 261-277.
(33) Cristobal, S.; Zemla, A.; Fischer, D.; Rychlewski, L.;
Elofsson, A. BMC Bioinf. 2001, 2, 5.
(34) Lill, M. A.; Danielson, M. L. J. Comput. Aided. Mol.
Des. 2011, 25, 13-19.
(35) Ribeiro, C.; Esteves da Silva, J. C. Photochem. Photo-
biol. Sci. 2008, 7, 1085-1090.
(36) da Silva, L. P.; da Silva, J. C. Photochem. Photobiol. Sci.
2011, 10, 1039-1045.
1
2
3
4
5
6
7
8
Ceronio, N.; Roa-de la Fuente, L. F.; Lobato-Garcia, C. E.
Bioorg. Med. Chem. Lett. 2013, 23, 5519-5522.
(4) Deeb, D.; Gao, X.; Jiang, H.; Arbab, A. S.; Dulchavsky,
S. A.; Gautam, S. C. Anticancer Res. 2010, 30, 3333-3339.
(5) Zhang, B.; Duan, D.; Ge, C.; Yao, J.; Liu, Y.; Li, X.; Fang,
J. J. Med. Chem. 2015, 58, 1795-1805.
(6) Ram, V. J.; Saxena, A. S.; Srivastava, S.; Chandra, S.
Bioorg. Med. Chem. Lett. 2000, 10, 2159-2161.
(7) Artese, A.; Costa, G.; Ortuso, F.; Parrotta, L.; Alcaro, S.
Molecules 2013, 18, 12051-12070.
(8) Thorne, N.; Inglese, J.; Auld, D. S. Chem. Biol. 2010, 17,
646-657.
(9) Li, J.; Chen, L.; Du, L.; Li, M. Chem. Soc. Rev. 2013, 42,
662-676.
(10) Fan, F.; Wood, K. V. Assay Drug Dev. Techn. 2007, 5,
127-136.
(11) Poutiainen, P. K.; Palvimo, J. J.; Hinkkanen, A. E.;
Valkonen, A.; Vaisanen, T. K.; Laatikainen, R.; Pulkkinen,
J. T. J. Med. Chem. 2013, 56, 1064-1073.
(12) Ueda, I. Anesthesiology 1965, 26, 603-606.
(13) Fraga, H.; Fernandes, D.; Fontes, R.; Esteves da Silva,
J. C. FEBS J. 2005, 272, 5206-5216.
(14) Bakhtiarova, A.; Taslimi, P.; Elliman, S. J.; Kosinski, P.
A.; Hubbard, B.; Kavana, M.; Kemp, D. M. Biochem. Bioph.
Res. Commun. 2006, 351, 481-484.
(15) Auld, D. S.; Thorne, N.; Maguire, W. F.; Inglese, J. P.
Natl. Acad. Sci USA 2009, 106, 3585-3590.
(16) Auld, D. S.; Lovell, S.; Thorne, N.; Lea, W. A.; Malo-
ney, D. J.; Shen, M.; Rai, G.; Battaile, K. P.; Thomas, C. J.;
Simeonov, A. P. Natl. Acad. Sci 2010, 107, 4878-4883.
(17) Fontes, R.; Fernandes, D.; Peralta, F.; Fraga, H.; Maio,
I.; Esteves da Silva, J. C. FEBS J. 2008, 275, 1500-1509.
(18) Zhang, H.; Bai, H.; Jiang, T.; Ma, Z.; Cheng, Y.; Zhou,
Y.; Du, L.; Li, M. Photochem. Photobiol. Sci. 2016, 15, 244-
249.
(19) Braeuning, A.; Vetter, S. Bioscience Rep. 2012, 32, 531-
537.
(20) Heitman, L. H.; van Veldhoven, J. P.; Zweemer, A. M.;
Ye, K.; Brussee, J.; AP, I. J. J. Med. Chem. 2008, 51, 4724-
4729.
(21) Bai, H.; Chen, W.; Wu, W.; Ma, Z.; Zhang, H.; Jiang,
T.; Zhang, T.; Zhou, Y.; Du, L.; Shen, Y.; Li, M. RSC Adv.
2015, 5, 63450-63457.
(22) Bai, H.; Zhu, P.; Wu, W.; Li, J.; Ma, Z.; Zhang, W.;
Cheng, Y.; Du, L.; Li, M. MedChemComm 2015, 6, 418-424.
(23) Bai, H.; Chen, W.; Wu, W.; Ma, Z.; Zhang, H.; Jiang,
T.; Zhang, T.; Zhou, Y.; Du, L.; Shen, Y.; Li, M. RSC Adv.
2015, 5, 63450-63457.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(37) Leitão, J. M.; da Silva, J. C. E. J. Photoch. Photobiol.
B.2010, 101, 1-8
(38) Wu, W.; Li, J.; Chen, L.; Ma, Z.; Zhang, W.; Liu, Z.;
Cheng, Y.; Du, L.; Li, M. Anal. Chem. 2014, 86, 9800-9806.
(39) Roda, A.; Guardigli, M.; Michelini, E.; Mirasoli, M.
TrAC Trac-Trend Anal. Chem. 2009, 28, 307-322.
(40) Hart, R. C.; Stempel, K. E.; Boyer, P. D.; Cormier, M.
J. Biochem. Bioph. Res. Commun.1978, 81, 980-986.
(24) Huang, S.-T.; Hsei, I.-J.; Chen, C. Bioorg. Med. Chem.
Lett. 2006, 14, 6106-6119.
(25) Ley, S. V.; Thomas, A. W. Angew. Chem. Int. Ed. 2003,
42, 5400-5449.
(26) Otwinowski, Z.; Minor, W. In Method Enzymol; Aca-
demic Press, 1997, pp 307-326.
(27) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.;
Winn, M. D.; Storoni, L. C.; Read, R. J. J Appl Crystallogr.
2007, 40, 658-674.
ACS Paragon Plus Environment

Page 9 of 9
Analytical Chemistry
1
2
3
4
5
6
For TOC Only
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment