Synthesis and biological evaluation of a series of aryl triazoles as firefly luciferase inhibitors

Article abstract of DOI:10.1039/c4md00368c

As the most studied bioluminescent system, firefly luciferase is widely applied in many aspects, such as developing small molecule probes, bioluminescent imaging, high-throughput screening, dual luciferase reporters, etc. Considering that a false positive

Full text of DOI:10.1039/c4md00368c

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Synthesis and biological evaluation of a series of
aryl triazoles as firefly luciferase inhibitors†
Cite this: DOI: 10.1039/c4md00368c
Haixiu Bai,a Peng Zhu,a Wenxiao Wu,a Jing Li,a Zhao Ma,a Wei Zhang,a Yanna Cheng,b
a
a
*
Lupei Du and Minyong Li
As the most studied bioluminescent system, firefly luciferase is widely applied in many aspects, such as
developing small molecule probes, bioluminescent imaging, high-throughput screening, dual luciferase
reporters, etc. Considering that a false positive phenomenon often emerges while researchers conduct
high-throughput screening based on firefly luciferase, and that the triazole core is a “privileged” scaffold
in drug design and development, we herein report a series of triazoles with potent inhibitory activity in
vitro and in vivo, comparable to that of the well-known inhibitor resveratrol. More interesting, a kinetics
study disclosed that these triazoles exhibited a brand new inhibition mode, mixed noncompetitive for the
substrate aminoluciferin and noncompetitive for ATP. Henceforth, these compounds can notify
researchers for possible “false positives”. Moreover, they will shed light on luciferase structure–function
mechanistic exploration and help expand its application in various areas.
Received 26th August 2014
Accepted 30th September 2014
DOI: 10.1039/c4md00368c
www.rsc.org/medchemcomm
bioluminescence system can ensure its extensive application in
many areas, such as bioluminescent imaging, quantitative high-
Introduction
throughput screening (qHTS), luciferase reporter gene assay,
detection of ATP, etc.
Bioluminescence is the production and emission of visible light
by a chemical reaction within a living organism. It occurs widely
in marine vertebrates and invertebrates, as well as in some
insects, fungi, microorganisms and terrestrial invertebrates.
Firey luciferase, as the most studied bioluminescent enzyme,
can catalyze the oxidation of luciferin and emit yellow to green
lights upon a two-step reaction. In such a rey luciferin–
luciferase system, oxygen, ATP and magnesium ions are
necessary as co-factors. In the rst step, rey luciferase cata-
lyzes the reaction between luciferin and ATP to produce a
luciferin–adenylate conjugate, and then the conjugate
undergoes oxygenation and cyclization to form a dioxetanone
anion (DxÀ). 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, as
well as oxyluciferin (OLH), CO2 and AMP (Scheme 1).1 The
wavelength of the bioluminescent light can range from 530 nm
to 640 nm, depending on multiple intermolecular interactions
(mostly hydrogen-bonding, p–p stacking and electrostatic
interaction), polarity of the solvent, pH and the microenviron-
ment of the enzyme etc. The unique characteristics of the rey
Despite the prevalent applications, there are still a few
drawbacks and limitations in the luciferase–luciferin system.
One case is quantitative high-throughput screening (qHTS) assay
based on luciferase bioluminescence, which has become widely
used in chemical biology and drug discovery applications. In
2006, D. M. Kemp claimed that resveratrol can potently inhibit
rey luciferase activity with a Ki value of 2 mM, cautioning
researchers that a previous study about resveratrol showing the
therapeutic value in various elds might be fundamentally
awed since they all utilized the rey luciferase assay.2 These
interesting results somewhat explain the “false positives” that
stand out as “promising compounds” through the biolumines-
cence-based qHTS but turn out to be a false alert in further
bioassays. Ever since then, several research groups reported their
“false positives” with potent rey luciferase inhibitory activity,
in which most of the inhibitors are small molecules with rigid
structures, commonly containing a heterocycle moiety, such as
thiazole, imidazole, oxadiazole, or a pyridine ring.3–9
In the current article, we report a series of novel aryl triazole
derivatives (Table 1) with rey luciferase inhibitory activity
both in vitro and in vivo. It is well known that the triazole core
has been generally considered as a “privileged” scaffold for a
variety of drug candidates, including antivirals, antifungal
agents, H1/H2 histamine receptor blockers, cholinesterase
active agents, etc., and as a result, it has been widely used in
many therapeutic agents, such as uconazole and ribavirin. In a
high throughput screening that relies on rey luciferase
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
bDepartment of Pharmacology, School of Pharmacy, Shandong University, Jinan,
Shandong 250012, China
† Electronic supplementary information (ESI) available: Experimental procedures
and 1H-NMR and HR-MS spectra. See DOI: 10.1039/c4md00368c
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Scheme 1 Mechanism of firefly bioluminescence.
reporter-gene assay, small molecular inhibitors of rey lucif- preliminary structure–activity relationship analysis, the triazole
erase, as these triazoles, are extremely likely to pop out as “hits”. structure is a valid inhibition core for rey luciferase. There-
Nevertheless, these “hits” could turn out to be “false positives” fore, identication of these rey luciferase inhibitors may help
when further evaluated with other bioassays. According to our to avoid futile efforts in the initial screening.
Table 1 Firefly luciferase enzymatic and cellular inhibition activity of novel triazolesa
Predicted
log P
Compounds
R1
Ar2
X
Enzymatic IC50a (mM)
Cellular IC50a (mM)
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
9a
9b
9c
9d
2,4-Dichloro
2-Methoxy
3-Methoxy
4-Methoxy
2,4-Dichloro
2-Methoxy
3-Methoxy
4-Methoxy
2,4-Dichloro
2-Methoxy
3-Methoxy
4-Methoxy
2,4-Dichloro
3-Methoxy
2,4-Dichloro
3-Methoxy
4-Chlorphenyl
4-Chlorphenyl
4-Chlorphenyl
4-Chlorphenyl
3-Pyridinyl
3-Pyridinyl
3-Pyridinyl
3-Pyridinyl
2-Furanyl
2-Furanyl
2-Furanyl
2-Furanyl
Phenyl
O
O
O
O
O
O
O
O
O
O
O
O
H,H
H,H
H,H
H,H
3.28 Æ 0.43
>100
>200
>200
4.61
3.37
3.37
3.37
2.72
1.47
1.47
1.47
2.67
1.43
1.43
1.43
4.62
3.38
4.78
3.53
22.3 Æ 1.10
55.6 Æ 4.02
>100
>200
6.65 Æ 0.78
>100
>200
>200
>200
>200
>200
>200
>200
>200
33.2 Æ 2.52
>100
47.9 Æ 2.26
>100
56.6 Æ 2.44
>100
53.3 Æ 3.25
>100
>200
Phenyl
2-Fluorophenyl
2-Fluorophenyl
190 Æ 3.90
44.1 Æ 4.72
>200
>100
102 Æ 3.74
11
>100
>200
4.93
Resveratrol
2.27 Æ 0.12
30.1 Æ 3.21
3.06
a
Assays were performed in triplicate (n $ 3); the values are shown as mean Æ SD.
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Moreover, considering that some of our triazole compounds salt upon reaction with NaNO2 under acidic conditions, and
demonstrated good inhibitory potency in vitro, in cellulo and in then followed by substitution with NaN3. Substituted N-benzyl-
vivo, they may be applied as quenching reagents in rey 2-propynyl amines 3a–c were easily prepared from aromatic
luciferase-based assays, such as dual luciferase reporter assays. acids 1a–c through chloridization and amidation in an ice-salt
These compounds' inhibition modes through kinetics assay bath. Intermediates 7a–b were conveniently prepared from
were well examined as well. In brief, these triazoles exhibited a benzyl bromides 6a–b through nucleophilic substitution with 2-
novel inhibition mode compared to those inhibitors reported propynylamine in an ice-salt bath.
before. The inhibition is mixed noncompetitive for the
Compound 11 was synthesized starting from 2,4-dichlor-
substrate luciferin while noncompetitive for ATP. This indi- oaniline in three steps. First of all, 2,4-dichloroaniline turned
cated that besides competing with luciferin for the active site, into diazonium salt upon reaction with NaNO2 under acidic
these triazoles might have yet another way to inhibit the conditions, then the diazonium salts reacted with furfuryl-
bioluminescence. Also, since many triazoles are reported as free amine through the catalyst CuCl2 by forming (5-(2,4-dichlor-
radical scavengers,10,11 and the intermediate in bioluminescent ophenyl)furan-2-yl) methanamine 10. Finally, compound 11 was
luciferin–luciferase is of the dioxetanone anion radical form, we produced by compound 10 reacting with 4-chlorobenzoyl chlo-
examined if our triazole inhibitors could quench the biolumi- ride using triethylamine as the base in tetrahydrofuran in an ice
nescence through the DPPH free radical scavenging assay. bath.
Hopefully, our efforts on biological activity and mechanism
search will help to further investigate luciferase-catalyzed
systems and further expand their application in many areas.
Biological evaluation
Inhibitory assay in vitro. In the preliminary screening, all
compounds were evaluated for their inhibitory activity on
puried recombinant rey (Photinus pyralis) luciferase. We
tested the inhibitory activity of increasing concentrations of the
Results and discussion
Chemistry
The synthesis of triazoles 8a–l, 9a–d and aryl furan compound compounds to get concentration–response curves (CRCs) to
11 is outlined in Scheme 2. In brief, triazole compounds 8a–l determine their IC50 values. Fig. 1a shows the concentration–
and 9a–d were synthesized through Cu(I)-catalyzed azide–alkyne response curves (CRCs) of compounds with IC50 < 100 mM. As
click chemistry in a one-pot reaction from appropriate azido- shown in Table 1, most of the triazoles showed moderate to
benzenes 5a–d and substituted N-benzyl-2-propynyl amines good inhibitory activity. Among them, compound 8a showed
3a–c and 5a–b in a mixture solvent of tert-butyl alcohol and the best potency with an IC50 value of 3.28 mM, comparable to
water in the absence of light.12 Azidobenzenes 5a–d were that of the positive control resveratrol. Compound 11 with a
prepared from corresponding anilines, rst forming diazonium furan ring instead of a triazole ring was inactive.
Scheme 2 Synthesis of triazoles 8a–l and 9a–d. Reagents and conditions: (a) SOCl2, toluene, 90 ꢀC; (b) 3-aminopropyne, CH2Cl2, 0 ꢀC; (c) HCl–
NaNO2, H2O; NaN3; (d) 3-aminopropyne, 0 ꢀC; HCl–EtOH; (e) 5a–d; CuSO4$5H2O, sodium-L-ascorbate, EtOH, t-BuOH, H2O. Synthesis of
compound 11. Reagents and conditions: (f) HCl–NaNO2, 0 ꢀC; (g) CuCl2, furfurylamine, room temperature; (h) 4-chlorobenzoyl chloride 4a,
Et3N, 0 ꢀC.
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Fig. 1 (a) Concentration–response curves for active compounds in recombinant firefly luciferase inhibition assay; (b) bioluminescence imaging
of ES-2-Luc cells incubated with increasing concentrations of inhibitors. For each concentration, three wells in vertical are treated the same as
the parallel group. Representative graphs are chosen from one experiment performed in triplicate.
Since the bioluminescence-based assay is widely used in 2-methoxy (8b, 8f) or 4-methoxy (8d, 8h). (d) Replacement of the
cellulo and in vivo, we further evaluated their inhibitory activity aromatic ring Ar2 with a more electron rich ring, e.g. a foran ring
in ES-2-Luc cells (a human ovarian cancer cell line transfected (8i, 8j, 8k, 8l) will lead to a tremendous loss of potency. Corre-
with rey luciferase). We incubated increasing concentrations spondingly, introduction of an electron-withdrawing group in
of inhibitors with ES-2-Luc cells for 12 h, and then tested their Ar2 (9c) will help to improve potency compared to 9a.
bioluminescence intensity using an IVIS Kinetic (Caliper Life
Cell viability assay. To rule out the possibility that the
Sciences, USA) equipped with a cooled charge coupled device decrease in bioluminescence might also be caused by cell death
(CCD) camera for bioluminescence imaging. As we can see in through incubation with our compounds, we tested the ES-2-
Fig. 1b, for compounds 8c, 9c, 9d and 9b, their inhibitory Luc cell viability of all compounds aer 12 hour incubation with
activity remained in cellular. However, some of the compounds 500, 250, 125 and 62.5 mM of compounds 8c, 9c, 9d and 9b,
with ne inhibitory potency at the enzyme level, including the which showed good activity in cellulo. The cell viability was
most potent compound 8a, only showed negligible to none evaluated by using MTT assay according to the standard
potency in cellular (8i, 8e, 8k, 8g, 9a). The irrational lipo-hydro protocol. Fig. 2 revealed that cells suffered only slight damage at
partition coefficient log P might be the reason for the loss of 500 mM. As a result, our compounds did not cause any signi-
potency. We used Chembiodraw Ultra 12.0 soware to predict cant damage in cell viability at <250 mM concentrations.
log P of these compounds. As we can see in Table 1, for
In vivo inhibition assays. Subsequently, compound 8c, most
compounds that retained potency in cellulo, their predicted potent both at the enzyme level and in cellular was chosen for
log P ranged from 3.06 to 3.38, with only one exception 9c. inhibition assay in xenograed balb/c-nu male mice. To avoid
Nevertheless, for compounds that lost potency, their predicted the individual variation of mice, we tested the bioluminescence
log P turned out to be either larger than 4.0 (8a and 9a) or lower as the baseline by injecting 100 mL of luciferin (0.5 mM) intra-
than 2.8 (8i, 8e, 8k, 8g). Thus, we can ascribe the loss of potency peritoneally into the mouse on the rst day, and then gave the
to poor permeability in the cell membrane caused by the
inappropriate lipo-hydro partition coefficient.
The preliminary structure–activity relationship can be
summarized as follows: (a) compound 11 with a furan ring
instead of a triazole core completely lost its potency. This
indicates that the triazole core is essential for inhibitory
potency, changing it to a furan ring will lead to a thorough loss
of potency; (b) the amide linker between the aryl ring Ar2 and
the triazole core is crucial for inhibitory activity, probably, by
restricting the exibility of the molecule, which is in accordance
with the previously reported ndings. In this case, our
compounds 9a, 9c, 9b and 9d proved to be much less potent
than 8a, 8e, 8c and 8g; (c) introduction of an electron with-
drawing group at the phenyl ring is favourable. Compounds
with an electron withdrawing group like 2,4-dichloro (8a, 8e) or
3-methoxy (8c, 8g) proved to be much more potent than the
corresponding compounds with an electron donating group
Fig.
2 Viability of ES-2-Luc cells after incubation with various
concentrations of compounds.
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mouse 12 hours to metabolize luciferin. Aer that, we injected
25 mL of inhibitor 8c or resveratrol (5 mM) into the tumor and
waited for another 12 hours; then we injected luciferin intra-
peritoneally into the mouse to measure its bioluminescence
again.13 For the normal saline group, we injected an equivalent
amount of sterile saline instead of inhibitors as the blank
group. The sterile saline was prepared using 15% DMSO, as the
inhibitor group, to eliminate the inuence caused by DMSO.
Due to the 24 h growth of the tumor, we can see that the blank
normal saline group suffered an increase of 146.13% in the total
bioluminescence. Therefore, we calculated the inhibition rate
by comparing with the saline group. As shown in Fig. 3,
compound 8c displayed good inhibitory activity with 38.31%
inhibition compared to that of positive control resveratrol
57.66% inhibition. These interesting results suggest that our
compound 8c presented reasonable bioluminescence quench-
ing behavior in vivo. To avoid the articial inuence, for the
same mouse before and aer injection with inhibitors, we
calculated its bioluminescence intensity using ROIs of the same
size. For some of the mice that developed two tumors in close
proximity, we chose the bigger one for intratumor injection. As
shown in Fig. 3b–c, the bioluminescence signal of the bigger
tumor attenuated, while that of the small one changed a little.
We can assume that 25 mL inhibitor intratumorly injected tar-
Fig. 3 Bioluminescence imaging of inhibitory activity in xenografted
tumors in nude mice. (a) Bioluminescence imaging of normal saline as
the blank group. (b) Bioluminescence imaging of resveratrol inhibition
activity. (c) Bioluminescence imaging of compound 8c inhibition
activity. (d) Quantification of relative total flux. The representative
graphs are chosen from one experiment performed in triplicate. ROIs geting the bigger tumor did not reach the small one. Therefore,
were drawn over individual wells. The error bars are SD for triplicated
measurements.
we set the ROIs as circles that surrounded only the bigger tumor
Fig. 4 Kinetics of inhibition of luciferase by 8a. (a) Luciferin saturation assay with increasing concentrations of 8a (1, 5, 10, 20, 50 and 100 mM). (b)
A Lineweaver–Burk plot of data in (a). (c) ATP saturation assay with increasing concentrations of 8a (1, 5, 10, 20, 50 and 100 mM). (d) A Line-
weaver–Burk plot of data in (c). The lines of (a) and (c) are fitted to Michaelis–Menten assay using GraphPad Prism software. The Lineweaver–
Burk plots are estimated using GraphPad Prism software.
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Table 2 Estimated Vmax and Km of luciferin and ATP
Concentration (mM)
No inhibitor
1 mM
5 mM
10 mM
20 mM
50 mM
100 mM
Luciferina
ATPb
Vmax (Rlu sÀ1
)
)
97.9 Æ 2.78
0.98 Æ 0.09
120 Æ 2.81
37.6 Æ 3.52
86.7 Æ 1.21
3.75 Æ 0.33
104 Æ 0.85
31.1 Æ 1.08
78.2 Æ 5.43
12.8 Æ 1.74
102 Æ 1.16
30.3 Æ 0.84
73.5 Æ 10.13
26.8 Æ 4.79
97.7 Æ 1.53
31.0 Æ 0.38
65.5 Æ 11.23
47.2 Æ 8.80
88.5 Æ 2.28
32.4 Æ 2.38
63.5 Æ 5.91
111 Æ 21.5
74.5 Æ 5.86
34.7 Æ 3.36
43.5 Æ 16.2
222 Æ 21.3
59.1 Æ 0.45
37.3 Æ 3.89
c
c
Km (mM)
Vmax (Rlu sÀ1
c
c
Km (mM)
a
Dependence of luciferase activity on the concentration of luciferin at 1 mM ATP was determined in the absence (no inhibitor) or presence of 1 mM,
5 mM, 10 mM, 20 mM, 50 mM and 100 mM of compound 8a. b Dependence of luciferase activity on the concentration of ATP at 500 mM luciferin was
determined in the absence (no inhibitor) or presence of 1 mM, 5 mM, 10 mM, 20 mM, 50 mM and 100 mM of compound 8a. c Michaelis constant Vmax
and maximum rate Km were estimated with the Michaelis–Menten kinetics equation using GraphPad Prism soware. The values are shown by
means Æ SD of three independent assays performed in triplicate.
instead of the whole one. By comparison, the inhibitory effect is at the cellular level, comparable to the positive control resvera-
quite obvious.
trol. Our follow-up in vivo experimental results indicated that
Enzyme inhibition kinetics assay. We chose the most potent compound 8c has reasonable potency in mice, in which it can
inhibitor 8a to investigate its kinetic characteristics of inhibi- effectively inhibit the bioluminescence in Balb/c-nu male mice
tion. First of all, we xed the concentration of the ATP constant bearing ES-2-luc subcutaneous tumors even more potently than
at 1 mM, and tested the enzyme activity against increasing the control resveratrol. Moreover, a preliminary SAR exploration
concentrations of luciferin aer inhibition by compound 8a implies that the triazole core is a valid rey luciferase inhibitor
(Fig. 4a). Then, we xed the concentration of luciferin at 500 core. The kinetics study indicated a mixed noncompetitive
mM, and measured the enzyme activity in the same way for ATP mechanism for the substrate luciferin and a noncompetitive
also (Fig. 4c). By the Lineweaver–Burk plot (Fig. 4b and d), we mechanism for ATP. As the triazole core is a “privileged” scaffold
get the Michaelis–Menten parameters Km and Vmax to evaluate for a variety of drug candidates, this article may notify
the inhibition mode for luciferin (Table 2). The inhibition of researchers to pay attention to “false positives” that might
enzyme by 8a caused a signicant increase in Km and a decrease emerge in qHTS. Moreover, the brand new inhibition mode of
in Vmax of luciferin in a dose-dependent way. This indicates a these triazoles can be helpful for further investigation into the
typical mixed noncompetitive inhibition, suggesting that the amazing luciferin–luciferase system and might help to expand
inhibitor not only binds to the active site of luciferase to its application in various areas since our compounds exhibited
compete with aminoluciferin, but also might have some other considerable potency in cellulo and in vivo.
way to quench the bioluminescence, possibly by interfering
with the luciferase–luciferin complex or the aminoluciferin–
adenylate conjugate. Another possibility is that the inhibitor
Acknowledgements
This work was supported by grants from the National Program
on Key Basic Research Project (no. 2013CB734000), the National
Natural Science Foundation of China (no. 81370085), the
Program of New Century Excellent Talents in University (no.
NCET-11-0306), the Shandong Natural Science Foundation (no.
JQ201019) and the Independent Innovation Foundation of
Shandong University, IIFSDU (no. 2014JC008 and 2012JC002).
can also bind to an allosteric site in luciferase distorting the
active site to a nonoptimal conformation for aminoluciferin,
thus resulting in a decrease in Vmax. Since some triazoles are
reported to be free radical scavengers, in the case of ATP, the
Vmax obviously decreased while the Km remained almost unal-
tered in a dose-dependent manner. This indicates that the
inhibition mode for ATP is noncompetitive. Thus, we are quite
sure that the inhibitor will not interfere with the amino-
luciferin–adenylate conjugate. For now, most of the reported
inhibitors are competitive or noncompetitive with respect to
luciferin. The triazole inhibitors in our study exhibited a new
inhibition mode.
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