Discovery of a series of 2-phenylnaphthalenes as firefly luciferase inhibitors

Article abstract of DOI:10.1039/c5ra12886b

As the most convenient and efficient bioluminescence system, the firefly luciferase/luciferin complex has been widely used in life science research and high-throughput screening (HTS). Nonetheless, the interpretation of firefly luciferase-based assay data

Full text of DOI:10.1039/c5ra12886b

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Discovery of a series of 2-phenylnaphthalenes as
firefly luciferase inhibitors†
Cite this: RSC Adv., 2015, 5, 63450
Haixiu Bai,‡ad Wang Chen,‡b Wenxiao Wu,a Zhao Ma,a Huateng Zhang,a Tianyu Jiang,a
a
c
a
b
a
*
*
Tianchao Zhang, Yubin Zhou, Lupei Du, Yuemao Shen and Minyong Li
As the most convenient and efficient bioluminescence system, the firefly luciferase/luciferin complex has
been widely used in life science research and high-throughput screening (HTS). Nonetheless, the
interpretation of firefly luciferase-based assay data is often complicated by the occurrence of “false
positives,” in part because firefly luciferase (Fluc) is subject to direct inhibition by HTS compounds that
might inadvertently act as inhibitors of its catalytic site. Here we report a series of 2-phenylnaphthalenes
as Fluc inhibitors with suitable potency both in vitro and in vivo. Besides, our compound 5 showed
significant systemic inhibition in transgenic mice. Enzymatic kinetics study reveals that compound 5 is
competitive for substrate aminoluciferin and noncompetitive for the second substrate ATP. Furthermore,
compound 5 exhibited good performance as a quenching agent in a dual-luciferase reporter assay. We
anticipate that these Fluc inhibitors will contribute to the broader utilization of bioluminescence in life
science research while circumventing or at least reducing the number of “false positives”.
Received 2nd July 2015
Accepted 8th July 2015
DOI: 10.1039/c5ra12886b
www.rsc.org/advances
luciferase catalyzes the reaction between luciferin and ATP,
leading to formation of luciferin–adenylate conjugate, and then
Introduction
the conjugate undergoes oxygenation, cyclization and forms
dioxetanone anion (Dxꢀ). Subsequently, the excited singlet state
of OL [1(OL)*], a light emitter intermediate is generated. Upon
the excited state 1(OL)* decay to the ground state oxyluciferin
(OLH), a yellow to green bioluminescent light is produced.
Firey oxyluciferin (OLH), CO2 and AMP are released at the
same time. The glowing process can be inhibited by the two
major products of the reaction, OLH and dehydroluciferyl-
adenylate (L-AMP), which lead to the ash prole of rey
bioluminescence. Joaquim C. G. Esteves da Silva et al. demon-
strated that OLH is a competitive inhibitor of luciferase (Ki ¼
0.50 ꢁ 0.03 mM) while L-AMP act as a tight-binding competitive
inhibitor (Ki ¼ 3.8 ꢁ 0.7 nM).2 Besides, CoA can stabilize the
light emission through thiolytic reaction between CoA and L-
AMP, which gives rise to dehydroluciferyl-CoA (L-CoA), a
much less powerful inhibitor.3,4 Firey luciferase can also
catalyze the synthesis of H2O2 from the same substrates when D-
LH2-AMP is oxidized into dehydroluciferyl adenylate (L-AMP).5
The emission wavelength can vary from 530 to 640 nm,
depending on parameters such as multiple intermolecular
interactions (mostly hydrogen bonding, p–p stacking and elec-
trostatic interaction), pH, solvent polarity, and the microenvi-
ronment of the enzyme. Luciferin, as the natural substrate of
Fluc, can emit normal yellow to green light with a peak wave-
length of 562 nm at neutral or alkaline pH, and red light peaked
around 614 nm at acidic pH with low intensity and quantum
yield. In 1966, White and McElroy claimed a modied substrate
aminoluciferin (Scheme 1) with red-shied bioluminescence
Bioluminescence is the production and emission of light by a
living organism, through natural enzyme-catalytic reactions. It
occurs extensively in marine vertebrates and invertebrates, as
well as in some fungi and microorganisms, including a number
of bioluminescent bacteria and terrestrial invertebrates such as
reies. Compared to chemiluminescence, the unique enzyme
catalytic mechanism of bioluminescence makes it more
convenient and efficient for detection, quantication, and
application. Given that bioluminescence displays strong speci-
city, high sensitivity and has no background interference in
bioassays, it has been widely used in biomedical, pharmaceu-
tical, bioanalytical and bioimaging applications. Fluc is the
most widely-used bioluminescence system up till now by cata-
lyzing the oxidation of luciferin and emitting yellow to green
lights with oxygen, ATP and magnesium ion acting as indis-
pensable cofactors (Scheme 1).1 In the rst step, rey
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE),
School of Pharmacy, Shandong University, Jinan, Shandong 250012, China. E-mail:
mli@sdu.edu.cn; Fax: +86-531-8838-2076; Tel: +86-531-8838-2076
bDepartment of Natural Products, Key Laboratory of Chemical Biology (MOE), School
of Pharmacy, Shandong University, Jinan, Shandong 250012, China. E-mail: yshen@
sdu.edu.cn; Fax: +86-531-8838-2108; Tel: +86-531-8838-2108
cCenter for Translational Cancer Research, Institute of Biosciences & Technology,
Texas A&M University Health Science Center, Houston, TX 77030, USA
dShandong Medicine Technician College, Taian, Shandong 271016, China
† Electronic supplementary information (ESI) available: 1H NMR, 13C NMR and
HPLC spectra for all compounds; absorbance spectra of compound 5. See DOI:
10.1039/c5ra12886b
‡ These authors contributed equally.
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(596 nm) and a higher affinity than natural luciferin.6 Up to now, phenylbenzothiazole,18 pyrrolo[2,3-b]quinoxalines,19 and aryl
luciferin and aminoluciferin are still the only two commonly triazoles20) were also reported, along with their modes of action,
used substrates in rey luciferase research. The long wavelength mechanisms and characteristics. In addition, some general
is the advantage of Fluc application in bioluminescent imaging anesthetics and alkanes are also reported to be Fluc inhibi-
since it can penetrate into tissues up to 3–4 cm. This unique tors.21–23 Fluc inhibitors and relative inhibition characteristics
characteristic of the Fluc system enables its exceptionally func- have been target for “false positives” discussion.24,25 As biolu-
tional applications both in vitro and in vivo, such as biolumi- minescence based reporter assays being more and more
nescent imaging, quantitative high throughput screening, frequently used, Fluc inhibition and the modes of inhibition
luciferase reporter gene assay, diagnostic applications, and (MOI) raised extensive interests. Auld et al. reported novel MOI
environmental monitoring.
of ataluren through the formation of multisubstrate adduct
Despite its enormous contribution to molecular biology and inhibitor (MAI), and further investigated the vital effect of its m-
pharmaceutical research, Fluc occasionally misleads our carboxylate on inhibitory potency.13 They also demonstrated
research directions to “false positives,” especially in high- that the inhibitory activity can be relieved by free coenzyme A
throughput screenings that rely on luciferase reporter assays. through promoting the thiolysis and dissociation of the MAI.
In 1965, Ueda et al. reported that general anesthetics inhibited Thorne et al. conducted a screening for Fluc inhibitors in NIH
the bioluminescence of puried rey luciferase.7 This Molecular Libraries Small Molecule Repository among the
phenomenon raised extensive research on external factors that 360 864 compounds, and found that more than 10% showed
might inuence the luminescence process, since that might inhibitory potency against Fluc.26,27 Their structure–activity
reduce the accuracy of bioassays. In 1998, Issaku Ueda et al. relationship analysis revealed that most of the inhibitors share
proved that fatty acids are specic inhibitors of rey luciferase similar scaffold, mostly being small, linear, and planar,
in competition with luciferin in micromolar ranges.8,9 In 2003, commonly containing heterocyclic rings, e.g. thiazole, imid-
Neil D Perkins et al. observed a strong inhibition of reporter azole, oxadiazole, or pyridine ring. Albeit the inhibitors caused
plasmids containing the rey luciferase gene while investi- “false positives” trouble storm, they can be utilized in a dual-
gating the effects of pithrin-a on the transcriptional activity of luciferase reporter assay kit (DLR, Promega Corporation, Wis-
NF-kB, pithrin-a was proven to be inhibitor of rey luciferase consin, USA). In the DLR Assay, the activities of rey and
both in vivo and in vitro.10 In 2006, Daniel M. Kemp et al. renilla luciferases are measured sequentially in a single sample.
claimed that resveratrol (structure shown in Scheme 1) can First, rey luciferase reporter is measured by adding substrate
potently inhibit rey luciferase activity with an IC50 value of 2 luciferin to generate a luminescent signal. Aer quantifying the
mM, cautioning that some studies on resveratrol might be rey luminescence, the bioluminescence is quenched, and the
fundamentally awed if based on luciferase reporter assays.11 renilla luciferase luminescence is initiated simultaneously by
Ever since then, several more research groups suffered Fluc adding Stop & Glo Reagent to the same sample. Thus, Fluc
inhibitors oriented “false positives” in luciferase reporter gene- inhibitors with good potency and high efficiency can be utilized
based assays. These Fluc inhibitors varied in structures, as the quenching agents. Besides, Pekka K. Poutiainen et al.
including the resveratrol structurally related NF-kB inhibitor evaluated Fluc inhibitors as a versatile tool for real-time moni-
(E)-2-uoro-40-methoxystilbene,12 the drug candidate ataluren,13 toring cellular uptake and trafficking of biomolecules.28
N-(quinolin-2-yl) benzamides,14 pyrrolo[2,3-d]pyrimidine
2-Phenylnaphthalenes are the scaffold of a marketed acne
analogues,15 5-benzyl-3-phenyl-4,5-dihydroisoxazoles and 5- treatment medicine adapalene (differin),29 and can be
benzyl-3-phenyl-1,4,2-dioxazoles,16 natural compound with frequently found in pharmaceuticals, natural products, and
quinazolin-4(3H)-one core.17 Some more Fluc inhibitors (e.g., 2- agrochemicals.30–33 This scaffold was also reported as a prom-
ising candidate for the treatment of cancer by targeting top-
oisomerase.34–37 Moreover, 2-phenylnaphthalenes were reported
to enhance estrogen receptor selectivity, thus may be thera-
peutically useful in treating certain chronic inammatory
diseases.38 Hence, here in this paper we report a series of 2-
phenylnaphthalenes as small molecule inhibitors of rey
luciferase to alert researchers of possible “false positives”. We
used resveratrol, a well-known potent rey luciferase inhibitor,
as the positive control to evaluate the activity of our compounds.
The most potent compound 2-(4-(6-methoxynaphthalen-2-yl)
phenyl)acetic acid (compound 5) inhibited rey luciferase
with IC50 value of 0.13 mM in vitro and 10.8 mM in cellulo, which
exceeded resveratrol in similar assays (2.38 mM in vitro and 29.6
mM in cellulo). Further in vivo assay of compound 5 (200 mM)
showed 54.1% inhibition in xenograed balb/c-nu male mice by
intratumor injection and 23.8% inhibition in CAG-luc-eGFP
L2G85 transgenic mice by tail intravenous injection. To
examine its kinetic features of inhibition, we conducted an
Scheme 1 Mechanism of firefly bioluminescence and structure of
D-luciferin, D-aminoluciferin, and resveratrol.
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enzyme kinetics assay to obtain Km and Vmax values under
varying concentrations of inhibitor compound 5. Compared to
the two major products of the reaction, OLH and L-AMP, which
lead to the ash prole of rey bioluminescence by competi-
tive inhibition (Ki ¼ 0.50 ꢁ 0.03 mM) and tight-binding
competitive inhibitor (Ki ¼ 3.8 ꢁ 0.7 nM) respectively,2 our
compound 5 was competitive for substrate aminoluciferin while
noncompetitive for ATP. Compound 5 inhibited aminoluciferin
with a Ki value of 0.12 mM, more potency than OLH. In addition,
compound 5 could be utilized as a quenching agent in a dual-
luciferase reporter assay and surpassed the respective compo-
nent in the DLR commercial kit.
In vitro inhibition assays
We conducted a preliminary screening of 11 compounds. All
compounds were evaluated for their inhibitory activity on
QuantiLum recombinant rey luciferase using the initial
concentrations of 10 and 100 mM. Among them, ve
compounds showed more than 50% bioluminescence inhi-
bition under the concentration of 10 mM. The inhibitory
results are shown in Fig. 1A. To conrm their inhibitory
potency, we further used increasing concentrations of the
compounds from 1 nM to 100 mM to obtain the accurate
concentration–response curves (CRCs) to determine their IC50
values. Fig. 1B shows the concentration–response curves
(CRCs) of compounds with IC50 < 10 mM. Results are
summarized in Table 1: all of ve compounds showed
signicantly enhanced inhibitory potency when compared to
the positive control resveratrol. Among them, compound 5 is
the most potent with an IC50 value of 0.13 mM, approximately
20 fold more potent than the positive control resveratrol (IC50
¼ 2.38 mM). Next, we evaluated their inhibitory activity in
cellulo using ES-2-Fluc cells (a human ovarian cancer cell line
transfected with rey luciferase expressing gene). We incu-
bated increasing concentrations of inhibitors with ES-2-Fluc
cells for 12 h, and then tested their bioluminescence inten-
sity using a Caliper IVIS Kinetic in vivo optical imaging system
(Caliper Life Sciences, now PerkinElmer, USA) equipped with
a cooled charge-coupled device (CCD) camera for biolumi-
nescence imaging. The corresponding CRCs are shown in
Fig. 1C. As shown in Table 1, compound 5 exhibited high
potency with an IC50 value of 10.8 mM, about 3-fold more
potent than resveratrol (IC50 ¼ 29.6 mM). For compounds 7
Results and discussion
Chemistry
The synthesis route of 2-phenylnaphthalenes 4–14 is outlined in
Scheme 2 using similar methods and experimental conditions
published previously.35 In brief, we started from the commercially
available 2-bromo-6-methoxynaphthalene (1), and got 2-(6-
methoxynaphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
(2) in a very high yield through a palladium(0) catalyzed Suzuki
coupling reaction with bis(pinacolato)diboron according to the
method described by Takagi.39 Through Suzuki cross-coupling
reaction of 2-(6-methoxynaphthalen-2-yl)-4,4,5,5-tetramethyl-
1,3,2-dioxaborolane (2) with the corresponding bromobenzene
derivatives (3a–g) and hydrolysis of the ester bond, we got
compounds 4–13. Ether cleavage was performed using boron
tribromide to afford corresponding compounds 14. More
synthetic details can be found in the ESI.†
Scheme 2 Synthesis of 2-phenylnaphthalenes 4–14. Reagents and conditions: (a) bis(pinacolato)diboron, potassium acetate, PdCl2(dp.pf),
dioxane, 60 ꢂC, 6 h. (b) Potassium fluoride PdCl2(dppf), dioxane, 90 ꢂC, 12h. (c) NaOH, EtOH, reflux then HCl. (d) Boron tribromide, DCM, ꢀ78 ꢂC,
12 h.
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and 10, their cellular inhibitory activities are almost compa- 125 mM, 62.5 mM) for 12 h before the cell viability measured by
rable to resveratrol. For compounds 6 and 14, their inhibitory MTT method. Fig. 2 displayed that the compounds 5, 10 and 14
activities are less potent, possibly due to poor membrane were nontoxic to ES-2-Fluc cells when concentrations were
penetration ability. Structural–activity relationship (SAR) below 250 mM. Compounds 6 and 7 presented low toxicity at 250
analysis reveals that the carboxyl acid on the benzene ring is mM. Besides, the compounds only absorbed light below 320 nm
essential for maintaining a strong inhibitory activity. Substi- (absorbance spectra of compound 5 shown in ESI†), thus
tution of the benzene with hydroxyl group (e.g., compounds making it less likely to interfere with the visible light of rey
11 and 12) or amino group (e.g., compound 13) abrogated its luciferase bioluminescence.
inhibitory activity. Protection of the carboxyl acid with a
methyl ester (e.g., compounds 4, 6, 8 and 9) also led to
In vivo inhibition assay by intratumor injection
substantially reduced efficacy. The hydroxyl group on the
Since the bioluminescence-based assay is widely used both
naphthalene ring is not favorable, while introducing a methyl
in vitro and in vivo, we further evaluated inhibitory activity
group to protect the hydroxyl group (e.g., compound 7 vs.
of compound 5 in well-established mouse xenogra tumor
compound 14) can enhance the potency both in enzymatic
bioluminescence imaging models.40–42 To avoid the indi-
level and in cellular assay.
vidual variation of mouse, we tested the bioluminescence
To rule out the possibility that the observed inhibitory
signal of total ux (photons per s per cm2 per steradian)
potency in cellulo might be due to cytotoxicity, we conducted
by injecting aminoluciferin intraperitoneally into the
an MTT cell viability assay to evaluate the cytotoxicity of
mouse on the rst day, and set it as the calibration value.
our compounds. Briey, ES-2-Fluc cells were incubated with
Aerward, 12 h were le for the mouse to metabolize away
different concentrations of compounds (500 mM, 250 mM,
the aminoluciferin. Aer that, we injected inhibitors (200 mM
Fig. 1 (A) Recombinant firefly luciferase inhibition assay initial screening. Inhibition potencies of all the compounds at concentrations of 10 and
100 mM were evaluated in the initial screening. (B) Concentration–response curves for active compounds in recombinant firefly luciferase
inhibition assay; (C) CRCs for active compounds in cellulo; representative graphs are chosen from one experiment performed in triplicate.
Table 1 Firefly luciferase enzymatic and cellular inhibition activity of 2-phenylnaphthalenesa
General structure:
Compounds
R1
R2
R3
Enzymatic IC50 (mM)a
Cellular IC50 (mM)a
5
7
10
14
6
–CH3
–CH3
–CH3
–H
–CH2COOH
–OH
–COOH
–OH
–H
–COOH
–H
–COOH
–COOCH3
0.13 ꢁ 0.05
0.14 ꢁ 0.05
0.22 ꢁ 0.08
0.87 ꢁ 0.62
2.26 ꢁ 0.04
10.8 ꢁ 2.49
28.2 ꢁ 6.84
47.4 ꢁ 8.41
146 ꢁ 17.5
331 ꢁ 54.3
–CH3
–OH
Resveratrol
2.38 ꢁ 0.08
29.6 ꢁ 2.62
a
Assays were performed in triplicate (n $ 3); values are shown as mean ꢁ SD.
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remained in the mouse injected with compound 5, while
67.0% le in the mouse injected with resveratrol. Notably,
our compound 5 (200 mM) showed the inhibitory activity of
45.9% in vivo.
In vivo inhibition assay in transgenic mice by tail intravenous
injection
Although compound 5 showed 45.9% inhibition in xenogra
tumor in mouse, it is still not sure whether compound 5 can
exert systemic inhibition effects in the body. Therefore, we
further evaluated its potency in CAG-luc-eGFP L2G85 transgenic
mice by tail intravenous injection. The transgenic mice
harboring the CAG-luc-eGFP L2G85 transgene exhibit wide-
spread expression of rey luciferase directed by the CAG
promoter. As shown in Fig. 4, our compound 5 demonstrated
23.8% systemic inhibition while resveratrol exhibited only
13.3% inhibition. This evidence indicates that compound 5
displayed better inhibition behavior than resveratrol in vivo.
Fig. 2 The viability of ES-2-Fluc cells after incubation with various
concentrations of compounds.
in sterile normal saline) into the tumor and waited for
another 12 hours before measuring its bioluminescence
signal of the total ux (photons per s per cm2 per steradian)
again. Bioluminescence imaging of the mice before and aer
compound inhibition is shown in Fig. 3A. The relative activity
was calculated by bioluminescence total ux ratio of day 2
to day 1. For the normal saline group, we injected an equiv-
alent amount of sterile saline as a blank group. The rey
luciferase residual activity was displayed in Fig. 3B. Due to
the 24 h growth of the tumor, we can see that the blank
normal saline group suffered an increase of 135.2% in total
ux. Therefore, we calculated residual total ux percentage
by the ratio of comparing the relative activity of inhibition
group with the saline group, setting the saline group as
100%. As shown in Fig. 3B, only 54.1% bioluminescence
Kinetics assay
We chose the most potent compound 5 for further analysis on
kinetic parameters. The Michaelis–Menten kinetics parame-
ters Km and Vmax of aminoluciferin and ATP in the absence and
presence of increasing concentrations of compound 5 were
examined (Fig. 5). First of all, we xed the concentration of
ATP at its Km, and measured the enzyme activity against
increasing concentrations of aminoluciferin aer inhibition
by compound 5 (Fig. 5A). Then, we xed the concentration of
aminoluciferin at its Km, and measured the same way for ATP
(Fig. 5C). By Lineweaver–Burk plot (Fig. 5B and D), we get
Michaelis–Menten parameter Km and Vmax to analyze the
Fig. 3 (A) Representative bioluminescence imaging of inhibition in xenografted tumors in nude mice. Relative activity was calculated by ratio of
total flux of day 2 to day 1. (B) Quantification of residual total flux percentage, calculated by ratio of relative activity of inhibitors group to normal
saline group. Inhibition rate of resveratrol and compound 5 can be calculated by 100% ꢀ residual total flux (%), which means 45.9% and 23.0%
inhibition, respectively. ** <0.005, * <0.05 (t-test, calculated by GraphPad Prism software).
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Fig. 4 (A) Representative bioluminescence imaging of inhibition by compound 5 compared to normal saline in transgenic mice; (B) repre-
sentative bioluminescence imaging of inhibition compared to normal saline by resveratrol in transgenic mice; (C) quantification of inhibition rate
by compound 5 and resveratrol. *** <0.001 (t-test, calculated by GraphPad Prism software).
inhibition mode for aminoluciferin (Table 2). For substrate was measured immediately at wavelength 590 nm and 460 nm,
aminoluciferin, compound 5 caused a signicant increase in emitted by Fluc and Rluc, respectively. As shown in Fig. 6,
Km in a dose-dependent way, while the Vmax remains unaltered compound 5 eliminated about 97% of the rey luciferase
(Table 2), which usually arises from the inhibitor having an bioluminescence without signicant inuence on Rluc biolu-
affinity for the active site of an enzyme where the substrate minescence, while being compatible with Rluc substrate coe-
also binds. The substrate and inhibitor compete for access to lenterazine. For resveratrol, 5.64% Fluc bioluminescence
the enzyme's active site. For the second substrate ATP, the Km remained. Besides, resveratrol also eliminated 33.4% Rluc
value remained almost unaltered while Vmax obviously bioluminescence. Thus, compound 5 showed potential as
decreased in a dose-dependent manner. This phenomenon
reveals a noncompetitive inhibition mode for ATP. The Ki
value can be obtained from the IC50 values and kinetics data
using the Cheng–Prusoff equation. Compound 5 potently
inhibits rey luciferase with a Ki value of 0.12 mM. Kinetics
data in low inhibition concentration was shown in ESI (Fig. S1
and Table S1†).
Dual-luciferase reporter gene assay simulation
During the course of enzymatic inhibition assays, we found
that our compounds inhibited rey luciferase in an extraor-
dinarily rapid way. The compounds reached its uppermost
potency upon exposed to rey luciferase within 30 seconds.
Therefore, we designed a dual-luciferase reporter assay simu-
lation, and used resveratrol and our compound 5 as a
quenching agent. The assay requires a mixture of rey
Fig. 5 Kinetics of inhibition of luciferase by compound 5. (A) Amino-
luciferase (Fluc) and renilla luciferase (Rluc). First of all, rey luciferin saturation assay with increasing concentrations (6, 12, 25, 50,
100 and 200 mM); (B) a Lineweaver–Burk plot of data in (A); (C) ATP
saturation assay with increasing concentrations (6, 12, 25, 50, 100 and
200 mM); (D) a Lineweaver–Burk plot of data in (C). The lines of (A) and
(C) are fitted to Michaelis–Menten assay using GraphPad Prism 5
luciferase substrates aminoluciferin and ATP were added into
luciferase mixture to initiate the rey bioluminescence.
Then, a mixture of inhibitor (50 mM) and Rluc substrate coe-
lenterazine (5 mM) was added to quench rey luminescence
software. The Lineweaver–Burk plots are estimated using GraphPad
and initiate renilla bioluminescence. Bioluminescence of both Prism software.
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Table 2 Kinetic parameters Vmax and Km of substrate, aminoluciferin and ATP
Concentration (mM)
No inhibitor
6 mM
12 mM
25 mM
50 mM
100 mM
200 mM
Amino-luciferin
Vmaxa (Rlu sꢀ1
)
4611 ꢁ 100
5.06 ꢁ 0.33
N.D.c
5352 ꢁ 359
71.6 ꢁ 6.80
0.12
5459 ꢁ 12
126 ꢁ 5.60
0.12
6366 ꢁ 320
313 ꢁ 39.4
0.12
5691 ꢁ 416
392 ꢁ 57.8
0.12
4706 ꢁ 238
557 ꢁ 61.6
0.11
4824 ꢁ 148
919 ꢁ 20.4
0.11
a
Km (mM)
Kib
ATP
Vmaxa (Rlu sꢀ1
)
13 379 ꢁ 55
1374 ꢁ 34
793 ꢁ 59.4
426 ꢁ 8.35
221 ꢁ 40.0
160 ꢁ 13.6
20 ꢁ 14.6
a
Km (mM)
48.7 ꢁ 5.44
33.8 ꢁ 1.86
42.1 ꢁ 6.98
33.0 ꢁ 6.41
29.9 ꢁ 6.41
38.7 ꢁ 1.92
39.4 ꢁ 2.66
a
Michaelis constant Vmax and maximum rate Km were estimated with Michaelis–Menten kinetics equation using GraphPad Prism soware. Values
are showed by means ꢁ SD of three independent assays performed in duplicate. b Ki values were calculated by the Cheng–Prusoff equation.43 N.D.:
c
not determined since Ki value is a constant for inhibitors.
Fig. 6 Dual-luciferase reporter assay simulation. Residual activity was calculated by RLU ratio of residual Fluc to Fluc. ** <0.005, * <0.05 (t-test,
calculated by GraphPad Prism software).
promising Fluc quenching agent in the combined application combined application of the rey luciferase system with
of bioluminescence systems.
other bioluminescence systems, e.g., renilla luciferase.
Acknowledgements
Conclusion
This work was supported by grants from the National Program
on Key Basic Research Project (no. 2013CB734000), the
Program of New Century Excellent Talents in University (no.
NCET-11-0306), the Program for Changjiang Scholars and
Innovative Research Team in University (no. IRT13028), the
Shandong Natural Science Foundation (no. JQ201019) and the
Fundamental Research Funds of Shandong University (no.
2014JC008).
In summary, here we disclosed
a series of 2-phenyl-
naphthalenes compounds as rey luciferase inhibitors.
Compound 5 inhibited rey luciferase with an IC50 value of
0.13 mM in vitro and 10.8 mM in cellulo. Besides, kinetic assay
indicates our compound 5 is competitive inhibitor with Ki
value of 0.12 mM, more potent than OLH (Ki ¼ 0.50 mM). In
addition, compound 5 (200 mM) showed 45.9% inhibition
in vivo on well-established mouse xenogra tumor models
by intratumor injection. Besides, compound 5 showed 23.8%
inhibition in transgenic mice by tail intravenous injection,
indicating it can exert systemic inhibition. Surprisingly,
the compound could exert its inhibition in an extremely
rapid way upon exposure to rey luciferase, or even to the
glowing rey luciferase–luciferin mixture in a dual-reporter
assay. Notably, small molecules with the similar framework
are ubiquitous in various compounds libraries designed for
high throughput screening. These results raise cautions in
“false positives” for those researchers employing rey
luciferase-based quantitative high throughput screenings.
Additionally, the ultra-fast and potent inhibition of our
designed compounds can contribute to the crossed or
Notes and references
1 J. Li, L. Chen, L. Du and M. Li, Chem. Soc. Rev., 2013, 42, 662–
676.
2 C. Ribeiro and J. C. Esteves da Silva, Photochem. Photobiol.
Sci., 2008, 7, 1085–1090.
3 H. Fraga, D. Fernandes, R. Fontes and J. C. Esteves da Silva,
FEBS J., 2005, 272, 5206–5216.
4 L. P. da Silva and J. C. da Silva, Photochem. Photobiol. Sci.,
2011, 10, 1039–1045.
5 H. Fraga, D. Fernandes, J. Novotny, R. Fontes and
J. C. Esteves da Silva, ChemBioChem, 2006, 7, 929–935.
63456 | RSC Adv., 2015, 5, 63450–63457
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Paper
RSC Advances
˜
6 E. H. White, H. Woerther, H. H. Seliger and W. D. McElroy, J. 25 J. M. M. Leitao and J. C. G. Esteves da Silva, J. Photochem.
Am. Chem. Soc., 1966, 88, 2015–2018.
Photobiol., B, 2010, 101, 1–8.
7 I. Ueda, Anesthesiology, 1965, 26, 603–606.
8 I. Ueda and A. Suzuki, Biophys. J., 1998, 75, 1052–1057.
26 N. Thorne, M. Shen, W. A. Lea, A. Simeonov, S. Lovell,
D. S. Auld and J. Inglese, Chem. Biol., 2012, 19, 1060–1072.
9 H. Matsuki, A. Suzuki, H. Kamaya and I. Ueda, Biochim. 27 N. Thorne, J. Inglese and D. S. Auld, Chem. Biol., 2010, 17,
Biophys. Acta, 1999, 1426, 143–150. 646–657.
10 S. Rocha, K. J. Campbell, K. C. Roche and N. D. Perkins, BMC 28 P. K. Poutiainen, T. Ronkko, A. E. Hinkkanen, J. J. Palvimo,
Mol. Biol., 2003, 4, 9.
A. Narvanen, P. Turhanen, R. Laatikainen, J. Weisell and
11 A. Bakhtiarova, P. Taslimi, S. J. Elliman, P. A. Kosinski,
J. T. Pulkkinen, Bioconjugate Chem., 2014, 25, 4–10.
B. Hubbard, M. Kavana and D. M. Kemp, Biochem. Biophys. 29 S. Wang, L. Zhang, X. Ding, Y. Zhou, J. Wang, H. Jiang and
Res. Commun., 2006, 351, 481–484. H. Liu, J. Org. Chem., 2011, 76, 4514–4521.
12 A. Braeuning and S. Vetter, Biosci. Rep., 2012, 32(6), 531–537. 30 R. S. Ward, Nat. Prod. Rep., 1995, 12, 183–205.
13 D. S. Auld, S. Lovell, N. Thorne, W. A. Lea, D. J. Maloney, 31 I. Kock, D. Heber, M. Weide, U. Wolschendorf and
M. Shen, G. Rai, K. P. Battaile, C. J. Thomas, A. Simeonov,
R. P. Hanzlik and J. Inglese, Proc. Natl. Acad. Sci. U. S. A., 32 E. Conchon, F. Anizon, B. Aboab and M. Prudhomme, J. Med.
2010, 107, 4878–4883. Chem., 2007, 50, 4669–4680.
14 L. H. Heitman, J. P. van Veldhoven, A. M. Zweemer, K. Ye, 33 Y. Dong, Q. Shi, H. C. Pai, C. Y. Peng, S. L. Pan, C. M. Teng,
B. Clement, J. Med. Chem., 2005, 48, 2772–2777.
J. Brussee and A. P. IJzerman, J. Med. Chem., 2008, 51,
4724–4729.
15 Y. Liu, J. Fang, H. Cai, F. Xiao, K. Ding and Y. Hu, Bioorg.
Med. Chem., 2012, 20, 5473–5482.
K. Nakagawa-Goto, D. Yu, Y. N. Liu, P. C. Wu, K. F. Bastow,
S. L. Morris-Natschke, A. Brossi, J. Y. Lang, J. L. Hsu,
M. C. Hung, E. Y. Lee and K. H. Lee, J. Med. Chem., 2010,
53, 2299–2308.
16 P. K. Poutiainen, J. J. Palvimo, A. E. Hinkkanen, A. Valkonen, 34 Y. Shen, W. Chen, B. Zhao, H. Hao, Z. Li, C. Lu and Y. Shen,
T. K. Vaisanen, R. Laatikainen and J. T. Pulkkinen, J. Med.
Chem., 2013, 56, 1064–1073.
17 B. Somanadhan, C. Leong, S. R. Whitton, S. Ng, A. D. Buss
and M. S. Butler, J. Nat. Prod., 2011, 74, 1500–1502.
18 D. S. Auld, Y. Q. Zhang, N. T. Southall, G. Rai, M. Landsman,
Biochem. Biophys. Res. Commun., 2014, 453, 302–308.
35 W. Chen, Y. Shen, Z. Li, M. Zhang, C. Lu and Y. Shen, Eur. J.
Med. Chem., 2014, 86, 782–796.
36 L. He, H. X. Chang, T. C. Chou, N. Savaraj and C. C. Cheng,
Eur. J. Med. Chem., 2003, 38, 101–107.
J. MacLure, D. Langevin, C. J. Thomas, C. P. Austin and 37 C. C. Cheng, Q. Dong, D. F. Liu, Y. L. Luo, L. F. Liu,
J. Inglese, J. Med. Chem., 2009, 52, 1450–1458.
19 A. Nakhi, M. S. Rahman, R. Kishore, C. L. Meda, G. S. Deora,
A. Y. Chen, C. Yu, N. Savaraj and T. C. Chou, J. Med.
Chem., 1993, 36, 4108–4112.
K. V. Parsa and M. Pal, Bioorg. Med. Chem. Lett., 2012, 22, 38 R. E. Mewshaw, R. J. Edsall Jr, C. Yang, E. S. Manas, Z. B. Xu,
6433–6441.
R. A. Henderson, J. C. Keith Jr and H. A. Harris, J. Med.
Chem., 2005, 48, 3953–3979.
39 J. Takagi, K. Takahashi, T. Ishiyama and N. Miyaura, J. Am.
Chem. Soc., 2002, 124(27), 8001–8006.
40 J. Li, L. Chen, W. Wu, W. Zhang, Z. Ma, Y. Cheng, L. Du and
M. Li, Anal. Chem., 2014, 86, 2747–2751.
20 H. Bai, P. Zhu, W. Wu, J. Li, Z. Ma, W. Zhang, Y. Cheng, L. Du
and M. Li, MedChemComm, 2015, 6, 418–424.
21 K. Takehara, H. Kamaya and I. Ueda, Biochim. Biophys. Acta,
2005, 1721(1–3), 124–129.
22 M. Keyaerts, I. Remory, V. Caveliers, K. Breckpot, T. J. Bos,
J. Poelaert, A. Bossuyt and T. Lahoutte, PLoS One, 2012, 41 M. S. Evans, J. P. Chaurette, S. T. Adams Jr, G. R. Reddy,
7(1), e30061.
M. A. Paley, N. Aronin, J. A. Prescher and S. C. Miller, Nat.
Methods, 2014, 11(4), 393–395.
23 S. Shanmugasundararaj, S. Lehle, H. I. Yamodo,
S. S. Husain, C. Tseng, K. Nguyen, G. H. Addona and 42 W. Wu, J. Li, L. Chen, Z. Ma, W. Zhang, Z. Liu, Y. Cheng,
K. W. Miller, PLoS One, 2012, 7(1), e29854.
24 A. Braeuning, Arch. Toxicol., 2015, 89(1), 141–142.
L. Du and M. Li, Anal. Chem., 2014, 86, 9800–9806.
43 C. Yungchi and W. H. Prusoff, Biochem. Pharmacol., 1973, 22,
3099–3108.
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