Discovery of Novel 11-Triazole Substituted Benzofuro[3,2-b]quinolone Derivatives as c-myc G-Quadruplex Specific Stabilizers via Click Chemistry

Article abstract of DOI:10.1021/acs.jmedchem.7b00016

The specificity of nucleic acids' binders is crucial for developing this kind of drug, especially for novel G-quadruplexes' binders. Quindoline derivatives have been developed as G-quadruplex stabilizers with good interactive activities. In order to improve the selectivity and binding affinity of quindoline derivatives as c-myc G-quadruplex binding ligands, novel triazole containing benzofuroquinoline derivatives (T-BFQs) were designed and synthesized by using the 1,3-dipolar cycloaddition of a series of alkyne and azide building blocks. The selectivity toward c-myc G-quadruplex DNA of these novel T-BFQs was significantly improved, together with an obvious increase on binding affinity. Further cellular and in vivo experiments indicated that the T-BFQs showed inhibitory activity on tumor cells' proliferation, presumably through the down-regulation of transcription of c-myc gene. Our findings broadened the modification strategies of specific G-quadruplex stabilizers.

Full text of DOI:10.1021/acs.jmedchem.7b00016

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Article
Discovery of Novel 11-Triazole Substituted Benzofuro[3,2-b]quinolone
Derivatives as c-myc G-Quadruplex Specific Stabilizers via Click Chemistry
De-Ying Zeng, Guo-Tao Kuang, Shi-Ke Wang, Wang Peng, Shu-Ling Lin, Qi Zhang, Xiao-Xuan Su,
Ming-Hao Hu, Honggen Wang, Jia-Heng Tan, Zhi-Shu Huang, Lian-Quan Gu, and Tian-Miao Ou
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 17 May 2017
Downloaded from http://pubs.acs.org on May 18, 2017
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Discovery of Novel 11-Triazole Substituted Benzofuro[3,2-b]quinolone
Derivatives as c-myc G-Quadruplex Specific Stabilizers via Click
Chemistry
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DeꢀYing Zeng†, GuoꢀTao Kuang†, ShiꢀKe Wang, Wang Peng, ShuꢀLing Lin, Qi
Zhang, XiaoꢀXuan Su, MingꢀHao Hu, Honggen Wang, JiaꢀHeng Tan, ZhiꢀShu Huang,
LianꢀQuan Gu, TianꢀMiao Ou*
School of Pharmaceutical Sciences, Sun Yatꢀsen University, Guangzhou 510006,
People’s Republic of China.
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ABSTRACT
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The specificity of nucleic acids’ binders is crucial for developing this kind of
drugs, especially for novel Gꢀquadruplexes’ binders. Quindoline derivatives have
been developed as Gꢀquadruplex stabilizers with good interactive activities. In order
to improve the selectivity and binding affinity of quindoline derivatives as cꢀmyc
Gꢀquadruplex binding ligands, novel triazole containing benzofuroquinoline
derivatives (T-BFQs) were designed and synthesized by using the 1,3ꢀdipolar
cycloaddition of a series of alkyne and azide building blocks. The selectivity towards
cꢀmyc Gꢀquadruplex DNA of these novel T-BFQs was significantly improved,
together with an obvious increase on binding affinity. Further cellular and in vivo
experiments indicated that the T-BFQs showed inhibitory activity on tumor cells’
proliferation, and presumably through the downꢀregulation of transcription of cꢀmyc
gene. Our findings broadened the modification strategies of specific Gꢀquadruplex
stabilizers.
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KEYWORDS
cꢀmyc gene; Gꢀquadruplex; benzofuro[3,2ꢀb]quinolone derivatives; click chemistry;
antitumor.
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ꢀ
INTRODUCTION
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Guanineꢀrich DNA sequences are able to fold into fourꢀstranded secondary
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structures known as Gꢀquadruplexes which arise from guanine DNA bases
selfꢀassociation by Hoogsteen hydrogen bonds.1ꢀ2 The Gꢀquadruplex structures have
drawn extensive attention, as the putative quadruplex sequences (PQS) are
widespread throughout the human genome, notably in telomeres and the promoter
region of oncogenes such as cꢀkit and cꢀmyc.3ꢀ4 With great biological and therapeutic
significance, Gꢀquadruplexes are now perceived as promising drug targets.5
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cꢀmyc is an important protoꢀoncogene that plays a crucial role in cell growth,
cellular proliferation, and apoptosis. Aberrant overexpression of the cꢀmyc gene is
closely related to the occurrence and the development of various cancers.6 The
nuclease hypersensitivity element III1 (NHE III1), located at 142ꢀ115 bp upstream
from the P1 promoter of cꢀmyc gene, is a guanineꢀrich sequence that can form
intramolecular GꢀQUADRUPLEX structures.7ꢀ8 These intramolecular Gꢀquadruplex
structures can act as a repressor of the cꢀmyc transcription level.9ꢀ10 Specific
Gꢀquadruplex binders that can promote formation or enhance the stability of the
Gꢀquadraplex could, in turn, inhibit the transcription activity and downꢀregulate the
expression of cꢀmyc gene.11ꢀ12 Thus, the design of drugs specifically interacting at the
cꢀmyc Gꢀquadruplex could pave the way for the discovery of novel antiꢀcancer agents.
Several small molecules have been reported with the ability to interact with cꢀmyc
Gꢀquadruplex, including porphyrin analogs,11 berberine derivatives,13 quindoline
derivatives,12 and hydrazone derivatives.14
Quindoline derivatives have been developed as effective Gꢀquadruplexes’
ligands and optimized in several ways (Figure 1A).12, 15ꢀ21 The planar and aromatic
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scaffold of the quindoline provides good recognition for Gꢀquartet through πꢀπ
stacking interactions. In addition, the introduction of an amino side chain on the
11ꢀposition,18, 21 the introduction of a positive charge by methylation at the 5ꢀN
position of 7ꢀfluorinated quindoline derivatives,17 and the introduction of a peptidyl
group at the 11ꢀposition of the aromatic benzofuroquinoline scaffold,16 lead to
significant improvement of their binding affinity to Gꢀquadruplex. Although these
compounds show interactions with the Gꢀquadruplex in the cꢀmyc gene with certain
selectivity,12, 18, 22 the specificities of this kind of compounds still need to be
improved.
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According to the representative model of the quindolineꢀGꢀquadruplex complex
structure,22 the short protonated amino alkyl side chain attached to the quindoline
skeleton is barely able to reach the nearest groove. Therefore, the modification in the
present study is aim to solve this problem.
The heteroaromatic 1,4ꢀsubstituted 1,2,3ꢀtriazole ring system has attracted
extensive interest in drug design, owing to its wide application as a synthetic building
block endowed with pharmacological potential.23ꢀ24 A variety of triazoleꢀlinked
groups leads to a better selectivity.25ꢀ28 Therefore, we designed a series of 11ꢀtriazole
benzofuroquinoline derivatives (T-BFQ, Figure 1B) to enhance the affinity and
selectivity of these derivatives for targeting cꢀmyc Gꢀquadruplex structure. In addition,
we took advantage of the CuAAC click chemistry reaction for extending the diversity
of the substituents,29 including groups with varying properties and linker lengths
which comprise openꢀchain amino groups (series a), azoꢀheterocyclic rings (series a),
hydroxyl group (series b), phenyl group (series b), etc.
Panels of experiments were applied to evaluate the effects of these T-BFQ
derivatives. We found that the introduction of the triazole moiety could not only
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enhance their binding affinities, but also significantly improved the selectivity toward
cꢀmyc Gꢀquadruplex. The combined results of these studies led to a structureꢀactivity
relationship that amino substituents and a longer side chain length are important for
improvement of their specific interactions with the cꢀmyc Gꢀquadruplex. Further
biological experiments showed that the selected derivatives could repress the
proliferation of human lymphoma cells, interfere the cell cycle, and presumably
through the downꢀregulation of transcription of cꢀmyc gene.
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Figure 1. Modification strategies of the quindoline scaffold in previous studies (A)
and the present study (B).
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ꢀ
RESULTS AND DISCUSSION
Chemistry. The synthetic route for the compounds was shown in Scheme 1.
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The key intermediate of 11ꢀchloroquindoline 1 was prepared following the procedure
previously reported. 21 The azido side chains 3a-3n and 4a-4k were synthesized from
the commercially available corresponding halide side chains via an azidation reaction
with NaN3 or TMSN3 (Scheme S1). All azides were directly used for copper(I)
catalyzed cycloadditions without any further purification. The 1,3ꢀdipolar
cycloaddition was performed with an excess of azido reactants, and CuSO4 was used
as copper source and sodium ascorbate as reducing agent. Most of the click reactions
were completed within 12 h at 35 oC to give all the products (5a-5n and 6a-6m).
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Scheme 1. Synthesis of 11ꢀtriazole benzofuro[3,2ꢀb]ꢀquinoline derivatives 5a-n, and
o
6a-m. Reagents and conditions: (a) pꢀTsOH, 2ꢀPropargylamine, 120 C, 6 hours; (b)
Azides 3a-n and 4a-k, CuSO₄·5H₂O, sodium ascorbate, THF/H2O, 35 oC, 6ꢀ12 hours.
Thermodynamic Stabilization Abilities of the T-BFQ Derivative on the
c-myc G-Quadruplex. One of the important properties of a good Gꢀquadruplex
ligand should be the stabilizing ability on quadruplexꢀforming sequence. Fluorescence
resonance energy transfer (FRET) assay was used to investigate the stabilizing ability
of T-BFQ derivatives on the Gꢀquadruplex DNA in the cꢀmyc gene promoter. The
promoter Gꢀquadruplex sequence of cꢀmyc containing a FAM label at 5’ and a
TAMRA (carboxytetramethylrhodamine) label at 3’ (FPu22T, Table S1) was used. An
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oligonucleotide that formed hairpin duplex DNA structure (F10T, Table S1) with
similar dualꢀfluorescence labels was used as a nonꢀquadruplex control. A previously
reported quindoline derivative j (numbered as 7 in this paper) with the same
benzofuroquinoline scaffold (Figure 1A) was used as a reference compound. 18
The results of Tm (melting temperature) values from the FRET melting
experiments were shown in Table 1, and all the original melting curves were shown in
Figure S1 and S2. The reference compound 7 could increase the Tm value of FPu22T
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o
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by 16.0 C, and increase the Tm value of F10T by 2.6 C. Comparing with 7, most
T-BFQ derivatives in series a showed higher Tm on FPu22T while lower Tm on
ꢀ
ꢀ
F10T, which implied an improved selectivity of these new compounds. For example,
o
5a could increase the Tm value of FPu22T by 22.0 C, and increase the Tm value of
o
F10T by 1.2 C, indicating that the incorporation of the triazole moiety enhanced the
interaction of the ligand with the Gꢀquadruplex DNA. On the other hand, compounds
in series b showed diverse activities. Generally, most compounds in series b had
lower stabilization abilities on both the Gꢀquadruplex DNA and the hairpin DNA than
compound 7.
The structureꢀactivity relationships (SAR) could be concluded from the
stabilizing activity of the tested compounds toward cꢀmyc Gꢀquadruplex. In particular,
the introduction of in situ protonated terminal substituents (amino groups and
azoꢀheterocyclic rings) in series a remarkably increased their Gꢀquadruplex
stabilizing efficiency, such as compound 5a, 5c, 5i, and 5j. In addition, the
introduction of monohydroxyl group (6a and 6b) could also result in increased ꢁTm
values, while the introduction of polyhydroxyl groups (6jꢀ6m), and phenyl groups (6d
and 6e), lead to rather weak stabilizing ability which may attribute to their oversize
bulk. These results revealed that the terminal substituents of the side chain, especially
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basic amino groups, could significantly enhance their ability of stabilizing
Gꢀquadruplex, which was consistent with the previous findings.18 This was reasonable
because electrostatic interaction between the ligand side chain and the Gꢀquadruplex
structure was an important factor for the recognition process according to previous
researches.17, 21 Furthermore, with the same terminal basic substituent, compounds (5i,
5k, 5m, and 5n) containing longer side chains showed relatively stronger stabilizing
ability to the cꢀmyc Gꢀquadruplex than compounds (5b, 5d, 5f, and 5g) with shorter
side chains. This result reinforced our idea that the ligandꢀquadruplex interactions
would be enhanced by extending side chain to allow deeper penetration of the
terminal substituents into the Gꢀquadruplex grooves so that more groove space would
be exploited.
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To further characterize the selectivity of these T-BFQ derivatives, we picked
compounds 5a, 5h, 5l, 5n, and 7 to performed the competitive FRET experiments. In
this assay, a nonꢀfluorescent duplex DNA competitor ds26 (Table S1) was used as the
competitor in the system containing FPu22T. As shown in Figure 2A, in the presence
of excess hairpin competitor (5ꢀ25 folds excess), the ꢀTm values of the FPu22T was
slightly affected in all the T-BFQ derivatives, while dropped dramatically in
compound 7. These results further indicated that the T-BFQ derivatives could
specifically stabilize Gꢀquadruplex with no significant effect on duplex DNA.
Table 1. Changes of oligomer’s melting temperatures (ꢁTm) obtained
from the FRETꢀmelting assay.
ꢁTm a (oC)
FPu22T F10T
ꢁTm (oC)
FPu22T
Cpd.
Cpd.
F10T
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22.0
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0.0
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0.1
0.3
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0.1
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1.7
1.0
0.1
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0.1
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0.4
2.6
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5b
5c
5d
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5f
5h
5i
5j
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5k
5l
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14.0
10.3
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5n
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6g
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ꢁTm = Tm (DNA + ligand) ꢀ Tm (DNA). The concentrations of FPu22T and
F10T were 0.4 ꢂM, and the concentrations of compounds were 2.0 ꢂM. The melting
temperature of FPu22T in the absence of compounds was 46 oC.
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Figure 2. A. Competitive FRET results for T-BFQ derivatives 5a, 5h, 5l, and 5n
without and with 5ꢀfold (2 ꢂM), 10ꢀfold (4 ꢂM) or 25ꢀfold (10 ꢂM) excess of duplex
DNA competitor (ds26). The concentration of FPu22T was 0.4 ꢂM. B. Competition
dialysis results of T-BFQ derivatives 5a, 5h, and 5l, and the reference compound 7
binding to the cꢀmyc Gꢀquadruplex or other DNA oligomers. A 10 mM TrisꢀHCl
buffer (pH 7.4) containing 100 mM NaCl was used in the experiments.
Binding Affinities and Selectivity of T-BFQ Derivatives on c-myc
G-quadruplex. In order to investigate the binding affinities of the T-BFQ
derivatives with cꢀmyc Gꢀquadruplex, and their selectivity for quadruplex versus
duplex DNA, the surface plasmon resonance (SPR) experiment was employed.30 The
binding constants (KD values) for the binding of the T-BFQ derivatives to DNA were
determined using SPR with biotinylated cꢀmyc Gꢀquadruplex DNA Pu22 and
biotinylated duplex DNA (Table S1) attached to a streptavidinꢀcoated sensor chip. As
shown in Table 2, most of the T-BFQ derivatives could bind to the cꢀmyc
Gꢀquadruplex more tightly than that to the duplex DNA, and the ratio of
KDduplex/KDPu22 ranged from 3.26 to 42.5. Among them, the most significant compound
was 5n, which possessed a binding selectivity of 42.5 folds (KDduplex/KDPu22). At the
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same time, the reference compound 7 showed similar binding affinities with both the
quadruplex DNA and the duplex DNA (KDPu22, 4.60 ꢂM; KDduplex, 4.46; KDduplex/KD
Pu22
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= 0.97). These results demonstrated that the incorporation of the 11ꢀtriazle containing
side chains could give an improvement of the binding affinity and selectivity.
Table 2. Equilibrium binding constants (KD) determined by SPR assay.
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KD (ꢂM)
KD (ꢂM)
Pu22
KDduplex/KD
Pu22
KDduplex/KD
Cpd.
Cpd.
Pu22 Duplex
Pu22 Duplex
2.43
3.05
3.46
3.65
3.85
3.78
2.38
2.84
1.75
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NA
18.0
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NA
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2.74
3.06
1.87
NA
7.78
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31.4
63.6
21.9
79.5
NA
3.26
4.74
5.13
7.15
23.2
7.15
42.5
ꢀꢀ
5a
5b
5c
5d
5e
5f
5h
5i
5j
5k
5l
5m
5n
6h
6i
5g
6a
6b
6c
6d
6e
6f
NA
NA
ꢀꢀ
NA
NA
ꢀꢀ
6j
NA
ꢀꢀ
7.33
3.26
0.94
4.60
NA
ꢀꢀ
6k
6l
NA
NA
ꢀꢀ
37.9
8.83
4.46
11.6
9.39
0.97
2.51
NA
ꢀꢀ
6m
NA
NA
ꢀꢀ
6g
7
a NA: not available between compound and DNA; b ꢀꢀ: not comparable.
Similar SAR with the above data in FRET could still obtained in this section,
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that most of the T-BFQ derivatives with positively charged amino substituents (series
a) could specifically and strongly bind to the cꢀmyc Gꢀquadruplex DNA, and
compounds with phenyl, alkyl, and glucosyl groups (6c, 6d, 6e, 6g, 6h, 6i, and 6j)
could barely bind to both the cꢀmyc and duplex DNA. While the length of the side
chains could not show a significant effect on the KD value.
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Restricted by the measuring principle itself, the SPR experiment requires
immobilized nucleotide sequences on the sensor chip, which may affect the forming
and the structure of the Gꢀquadruplex. Thus, alternative methods that could measure
in an absolute solution environment should be applied to give binding data. Here,
microscale thermophoresis (MST) was further applied since it is a sensitive method
for quantitative analysis of molecular interactions in solution at the microliter scale.31
Data in Table 3 exhibited consistency with those in the SPR assay. Firstly, most of the
T-BFQ derivatives with positively charged amino substituents (series a) could
specifically and strongly bind to the cꢀmyc Gꢀquadruplex DNA. The calculated KD
values ranged from (0.3 ± 0.01 ꢂM to 29.1 ± 1.39 ꢂM). Among them, compound 5h
Pu22
showed strongest selectivity toward cꢀmyc Gꢀquadruplex (KDduplex/KD
= 35.0).
Speaking of the reference compound 7, it showed similar binding affinity and
selectivity with those in the SPR assay (KDPu22, 3.36 ± 0.24 ꢂM; KDduplex, 3.79 ± 0.09
Pu22
ꢂM; KDduplex/KD
= 1.13). Again, weak or notꢀdetectable binding effects were
observed on the derivatives with phenyl, alkyl, and glucosyl groups (6c, 6d, 6e, 6f,
and 6k).
In addition, competition dialysis measurements were performed to further verify
the selectivity of the compound. The cꢀmyc (Pu27) oligomer together with several
other oligomers (Table S1), including duplex DNA (hairpin), single strand (cꢀmycꢀmu
and dT30), and other Gꢀquadruplex oligomers (bclꢀ2, HꢀRas, cꢀkit, RET, VEGF, and
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HTGꢀ22) were placed in independent dialysis cassettes after the general annealing
process. All the dialysis cassettes were submerged in a solution containing
compounds and allowed to dialyze for 24 h. After spectrophotometric quantitation, the
TꢀBFQs compound 5a, 5h, and 5l were found to exhibit the highest binding with
cꢀmyc quadruplex, and weaker binding with other kinds of DNA structures (Figure
2B). As a reference, compound 7 showed similar binding affinity on cꢀmyc and
cꢀmycꢀmu or hairpin. Although we found the selectivity of the TꢀBFQs improved
comparing with compound 7, we need to clarify that this selectivity focused on the
binding between cꢀmyc quadruplex and duplex DNA. In fact, we could find that the
TꢀBFQs still bind to other kinds of quadruplex DNAs, just like compound 7.
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Table 3. Calculated binding constants (KD) determined by MST assay.
KD (ꢂM)
Pu22 Duplex
KD (ꢂM)
Pu22 Duplex
Pu22
KDduplex/KD
KDduplex/KD
Cpd.
Pu22Cpd.
2.3±0.19 50.7±2.18
1.8±0.12 24.7±0.66
29.1±1.39 10.1±0.588
2.4±0.09 54.7±2.10
1.8±0.15 31.2±0.55
6.3±0.44 113.0±3.34
22.0
13.7
0.35
22.8
17.3
17.9
11.4
4.5
0.3±0.01 10.5±0.13
4.2±0.21 13.2±0.78
1.3±0.06 11.0±0.35
4.3±0.13 33.7±0.76
1.1±0.10 29.5±1.17
5.9±0.18 23.3±0.40
35.0
3.14
8.5
5a
5b
5c
5d
5e
5f
5h
5i
5j
7.84
26.8
3.95
4.64
0.77
2.66
5k
5l
5m
5g 3.1±0.12 35.2±0.63
6a 2.8±0.08 12.6±0.93
5n 2.2±0.09 10.2±0.29
6h 35.4±2.57 27.3±2.54
2.1±0.12 3.4±0.13
1.6
99.2±13.3264.0±6.71
6i
6b
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NAa
NA
NA
NA
NA
NA
NA
ꢀꢀ b
ꢀꢀ
56.6±3.07398.0±7.42
7.03
ꢀꢀ
6c
6d
6e
6f
6j
6k
6l
NA
NA
NA
ꢀꢀ
6.4±0.30 12.5±0.97
5.5±0.45 30.0±2.74
3.36±0.24 3.79±0.09
1.95
5.45
1.13
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NA
ꢀꢀ
6m
233.0±14.0
ꢀꢀ
6g
7
a NA: not available between compound and DNA
；
b ꢀꢀ: not comparable
Interactions of the T-BFQ Derivatives on c-myc G-quadruplex. Basing
on all the data from above assays, the T-BFQ derivatives with in situ protonated
terminal substituents (amino groups and azoꢀheterocyclic rings) in series a
remarkably increased their interactions with the cꢀmyc Gꢀquadruplex than the
reference compound 7. While the introduction of monohydroxyl, phenyl, alkyl, and
glucosyl groups in series b could not interact well with the cꢀmyc Gꢀquadruplex. To
comprehensively evaluate the interactions of these new synthesized T-BFQ
derivatives, we selected compound 5a (with the highest ꢀTm value), 5h (sharing same
end group with 5a), 5n (exhibited the highest selectivity in the SPR assay), and 5l
(exhibited the highest selectivity in the MST assay) for further studies.
Circular dichroism (CD) spectroscopy is usually used to determine the
conformational of Gꢀquadruplex DNA and the effects of ligands binding on
quadruplex structures.30 The oligomer Pu22 (Table S1) was used as the cꢀmyc
Gꢀquadruplex forming sequence. The conformational properties of the Pu22
Gꢀquadruplex DNA induced by the selected T-BFQ derivatives (5a, 5h, 5l, and 5n)
and the reference compound 7 were monitored by the CD spectroscopy. As is shown
in Figure 3A, in the presence of 100 mM KCl, the Pu22 sequence showed a positive
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peak at 262 nm and a negative peak at 242 nm, which suggested a typical parallel
Gꢀquadruplex structure. Upon the addition of selected compounds to Pu22, no
significant change was observed while the peak height was slightly increased,
indicating that T-BFQ could still maintain parallel cꢀmyc Gꢀquadruplex conformation
in the presence of potassium ion.
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The CD spectroscopy of Pu22 in the absence of metal ion was also studied. As
shown in Figure 3B, similar with the CD spectrum of Pu22 in the presence of KCl,
the CD spectrum of Pu22 exhibited a negative peak at 240 nm and a positive peak at
260 nm. The positive peak at 260 nm and the negative peak at 240 nm were
drastically increased after the treatment with T-BFQ derivatives. In addition, more
significant changes were observed upon the addition of the T-BFQ derivatives than
the reference compound 7. All these CD studies illustrated that tested compounds
could effectively induce the formation of parallel cꢀmyc Gꢀquadruplex conformation
in the absence of metal cations, which further indicated a strong interaction between
the ligands and Gꢀquadruplex DNA.
Figure 3. CD spectra of Pu22 (5 ꢂM) in 10 mM TrisꢀHCl buffer (pH 7.4) in the
presence of 100 mM KCl (A), and in the absence of KCl (B). CD spectra of Pu22
(black line), and Pu22 in the presence of 5 ꢂM of 5a, 5h, 5l, 5n, and 7, respectively.
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Effects of the T-BFQ Derivatives on the Proliferation of Tumor Cells.
To investigate the antitumor activity of the derivatives, MTT assay was employed to
evaluate the cytotoxicity of all the T-BFQ derivatives against human lymphoma cell
lines Raji and CA46, and lung adenocarcinoma cell line A549, as well as primary
cultured mouse mesangial cell line (in which the proliferation does not depend on
cꢀmyc expression). The cytotoxicity of the T-BFQ derivatives were shown as IC50
values (concentration for 50% inhibition) in Table 4. As for the control, the reference
compound 7 showed strong cytotoxicity in all cells, including tumor cells and normal
cells. Most compounds in series a showed diverse cytotoxicity between tumor cells
and normal cells, indicating the specify of these compounds were improved. Among
them, compound 5a, 5c, 5e, 5f, 5h, 5j, 5l, and 5m, showed strong cytotoxicity against
Raji, A549, and CA46 cells, while weak cytotoxicity against primary cultured mouse
mesangial cells. However, most compounds had weak cytotoxicity in all cells, such as
compound 6j and 6l. Although with a few exceptions, the structureꢀactivity
relationship in MTT assays was basically consistent with that found in in vitro assays.
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Table 4. IC50 values (ꢂM) of derivatives against tumor cells and the primary cultured
mouse mesangial cell.
Mesangial
cells
Mesangial
cells
Raji
5.53
A549
3.83
CA46
Raji A549 CA46
Cpd.
Cpd.
1.06
0.54
21.87
48.03
10.75
>50
3.06 3.18 0.05
0.19 10.5 0.38
0.27 3.73 1.30
15.74
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18.32
0.10
2.80
>50
2.28
12.06
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15.79
22.02
18.00 20.64 12.76 16.58
5k
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2.93
2.73
5.27
5.11
0.53
2.74
5.60
19.53
19.84
25.24
28.27
47.94
>50
3.96 0.26 0.02
5m 4.91 5.15 0.17
16.05
30.45
20.81
41.27
5e
5f
5l
27.40
3.01
13.90 12.75 1.50
3.70 9.75 6.15
5g
6a
6b
6c
6d
6e
6f
5n
6h
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15.29 21.86
28.79 19.42
7.55
11.53
13.85
15.12
14.75
14.02
14.10
6i 5.60 12.12 10.64 42.18
5.10
4.67
>50
25.08
>50
>50 >50 >50
22.16 25.30 9.73
>50 >50 46.09
43.09 >50 >50
0.05 2.11 0.01
>50
34.92
>50
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16.25
>50
11.62
33.64 19.16
41.67 17.76
>50
>50
6m
7
6g
>50
5.51
To precisely identify the effects of the T-BFQ derivatives on cell’s proliferation,
we took the real time cellular analysis (RTCA) assays using compound 5a, 5h, 5l, 5n,
and the reference compound 7. A549 cells (with overexpression of cꢀmyc) and
primary cultured mouse mesangial cells (the proliferation does not depend on cꢀmyc
expression) were treated with various concentrations of compounds (0 ꢂM, 1/8 IC50,
1/2 IC50, and IC50) in RTCA for 100 h.
The cell proliferation results of 5a and 7 were shown in Figure 4 (results of 5h,
5l, and 5n were shown in Figure S3). Compound 5a showed significant growth arrest
on A549 cells at the concentration of 5 ꢂM, while no effect on primary cultured
mouse mesangial cells. To be noted, the inhibitory activity of 7 on A549 cells was
stronger than 5a, while the inhibitory activity could also be observed on normal cells
at high concentration. Similar data could be detected in 5h, 5l, and 5n (Figure S3).
These results were consistent with the MTT results that the derivatives had relatively
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selectivity on tumor cells over normal cells, which might come from their specificity
of cꢀmyc gene. To exclude the possibility that differential effects of the compounds
come from cellular uptake efficiency, we also performed the cellular uptake
experiments. The uptake amounts of compounds in the mesangial cells were higher
than those in A549 cells (Figure S4), indicating that compounds exhibited lower
cytotoxity on the mesangial cells wasn’t due to cellular uptake.
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Figure 4. The effect of compound 5a and 7 on the proliferation of A549 cells (A) and
primary cultured mouse mesangial cells (B) measuring by using RTCA assay. A549
lung adenocarcinoma cells and primary cultured mouse mesangial cells were seeded
(2000 cells each well) on EꢀPlate 16 PET culturing about 20 h before compound
treatment. Cells were treated with various concentrations of 5a, 7, or DMSO control.
Down-Regulation of c-myc Transcription via Specific Interaction with
the G-Quadruplex Forming Sequence in the Promoter. To investigate
whether the derivatives could affect the cꢀmyc transcription, dualꢀluciferase reporter
assay, reverseꢀtranscriptionꢀpolymerase chain reaction (RTꢀPCR), and Western blot
assay were firstly applied. Dualꢀluciferase reporter assays were used with a psicheck2
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plasmid carrying the cꢀmyc promoter in front of the Renilla luciferase (Figure 5A),
and the same plasmid carrying Firefly luciferase downstream. After transfection, Raji
cells were treated with the T-BFQ derivatives (at 1/2 IC50) for 48 h. As shown in
Figure 5B, the ratio of Renilla/Firefly luciferase with the cꢀmyc promoter decreased.
Most derivatives could cause more obvious decrease in the ratio than the reference
compound 7. To be noted, the structureꢀactivity relationship found in in vitro
experiments, that compounds containing an amino groups or an azoheterocyclic ring
showed stronger activities, could not be found in the dual luciferase reporter assay. All
derivatives showed similar inhibitory activities on luciferase transcription. The
possible reason might be the complicated mechanism of other compounds in cells.
To identify whether this inhibitory activity was due to the interaction with the
Gꢀquadruplex, we further constructed plasmid carrying the mutant cꢀmyc promoter
which could not form a Gꢀquadruplex structure. Four compounds, 5a, 5h, 5l, and 7
possessing relative strong activities in all the above assays, were used in this assay. As
shown in Figure 5C, these compounds showed significant inhibitory activities on
wildꢀtype plasmid, while no significant effects on mutant plasmid.
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Figure 5. The effects of T-BFQ derivatives on cꢀmyc promoter’s activity via dual
luciferase reporter assays. (A) Schematic diagram of the plasmids used in dual
luciferase reporter assays. The psiCHECK2 plasmid carrying the cꢀmyc promoter in
front of the Renilla luciferase, and the Firefly luciferase in the same plasmid, was
transfected into cells. (B) Column graph showing the relative expression level of the
Renillar luciferase (activity of Renilla luciferase/activity of Firefly luciferase) after
the addition of T-BFQ derivatives. All the experiments were repeated for three times.
(C) Column graph showing the relative expression level of the Renillar luciferase
(activity of Renilla luciferase/activity of Firefly luciferase) in plasmids containing
wildꢀtype or mutant constructs after the addition of T-BFQ derivatives. Cells were
treated with 1/8 IC50 (low), 1/2 IC50 (medium), and IC50 (high) of compounds. All the
experiments were repeated for three times.
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To further identify whether this downꢀregulation was happened through T-BFQ
derivatives’ interactions with the cꢀmyc Gꢀquadruplexꢀforming sequence, an exon
specific experiment was carried out. The translocation between chromosomes 8 and
14 in Burkitt’s lymphoma CA46 cell can produce varying MYC mRNAs.
Gꢀquadruplexꢀforming sequence can only exist in the fragment (8;14) of
nonꢀtranslocated chromosome (NT), but not in the fragment (14;8) in translocated
CA46 cells. Therefore, the NT products (mRNA products from exon 1ꢀ3) are normal,
with a functional MYC under the control of a Gꢀquadruplex; whereas the functional
MYC produced from the fragment (14;8) (mRNA products from exon 2 and 3) on the
T allele lacks Gꢀquadruplexꢀmediated control. Using primers specific to two different
exons, the exon 1 and exon 2 mRNA products, respectively, of the CA46 cell line can
be examined independently (Figure 6A).32ꢀ33 As shown in Figure 6B, the amplification
of exon 1 in CA46 and Ramos cells were decreased upon addition of three
compounds, 5a, 5h, and 5l, respectively; while no significant changes were observed
in the amplification of exon 2 in these two cells. As for the control, the reference 7
could decrease mRNA products for both the exon 1 and exon 2 in these two cells.
These results gave further evidence that the specificity of the new T-BFQ derivatives
were improved.
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Figure 6. Effects of T-BFQ derivatives on cꢀmyc’s translocation. (A) Exonꢀspecific
expression in Raji cells based on translocation events. (B) Realꢀtime RTꢀPCR results
of Exonꢀspecific expression in CA46 and Ramos cells upon addition of compound 5a,
5h, 5l, and 7 with concentrations from 0 to 2 ꢂM. mRNA relative expression was
calculated with ct values and columned in graph. All the experiments were repeated
for three times.
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Furthermore, we explored the effects of 5a, 5h and 5l on the mRNA level of
cꢀmyc, and subsequent the protein level. In Figure 7A, the results of RTꢀPCR revealed
that 5a, 5h and 5l reduced the mRNA level of cꢀmyc in a doseꢀdependent manner but
didn’t affect the mRNA level of βꢀactin. These results suggested that the compounds
5a, 5h and 5l could suppress the transcription of cꢀmyc and subsequently suppress the
translation of this gene (Figure 7B).
The overall evaluation results of exon specific experiment, RTꢀPCR and western
blot suggested that compound 5a, 5h and 5l, as stabilizer to cꢀmyc Gꢀquadruplex,
could specifically interact with cꢀmyc Gꢀquadruplexꢀforming sequence and hence
downꢀregulate cꢀmyc transcription in cancer cells.
To make a clear map of compounds’ selectivity in cell, we further evaluated the
transcription level of some other oncogenes (Figure S5), including
Gꢀquadruplexꢀrelated genes PDGFA, RET, cꢀkit, kꢀRas, and bclꢀ2, and other
oncogenes without Gꢀquadruplexꢀforming sequences (GLI1 and AML). It turned out
that the compounds didn’t show significant effects on most of these genes’
transcription. Moreover, we constructed dualꢀluciferase plasmids carrying different
Gꢀquadruplexꢀforming genes’ promoters (including bclꢀ2, RET, cꢀkit, and VEGF).
After detection of the luciferase expression, we found that these compounds had little
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effects on these gene’s promoter activity (Figure S6).
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Figure 7. The Effects of 5a, 5h and 5l on cꢀmyc transcription and expression after 6ꢀh
treatment in Raji cells. (A) The transcription level of cꢀmyc gene in the presence of 5a,
5h and 5l in Raji cells by using RTꢀPCR. βꢀactin was used as an internal control. All
the experiments were repeated for three times. (C) Expression level of CꢀMYC
protein under the treatment of increased concentrations of 5a, 5h and 5l in Raji cells
by using Western blot. βꢀActin was used as an internal control. All the experiments
were repeated for three times.
G0/G1 Phase Arrest of Raji Cells by T-BFQ Derivatives. Since cꢀmyc is
an important oncogene and is closely related with the cell cycle regulation, the three
compounds were further evaluated their effects on cell cycle of Raji cells by flow
cytometric assay which enables quantification of the total cellular population in
different phases of the cell cycle (G0/G1, S, and G2/M). These compounds showed
similar effects on cell cycle of Raji cells, which arrested cells in the G0/G1 phase with
an increase about 15% upon treatments with compounds at 1 ꢂM concentration for 6 h
(Figure 8). Specifically, the representative compound 5a increased cells in the G0/G1
phase from 32.6% to 48.4%, and correspondingly decrease cell population in the
G2/M phase.
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Figure 8. The induction of G0/G1 phase arrest in Raji cells by compounds 5a, 5h and
5l. (A) Cell cycle analysis after propidium iodide (PI) staining after 6ꢀh treatment
with 1 ꢂM 5a, 5h, and 5l, or 0.1% DMSO in Raji cells. (B) The percentage of cells in
different phases of the cell cycle, analyzed by EXPO32 ADC software. All the
experiments were repeated for three times.
T-BFQs Inhibit Tumor Growth in a Human Lung Cancer Xenograft.
Considering the importance of cꢀMyc for tumor growth, we evaluated compound 5l’s
antitumor activity in vivo. Firstly, a colony formation assay was performed to evaluate
whether the TꢀBFQ compounds reduced the tumorigenicity and vitality of A549 cells.
As shown in Figure S7, after treated with 5a, 5h, 5l, and 7 for 8 days, colony
formation obviously decreased.
In addition, the in vivo antiꢀtumor activities against a human lung cancer
xenograft (A549) in athymic nude mice was evaluated. The results were presented in
Figure 9. An experimental group of ten tumorꢀbearing mice was treated with
compound 5l once a day for 12 days at a dose of 6.25 mg/Kg. No significant change
in body weight was observed during the treatment (Figure 9A). The negative control
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group, which was treated with saline only, had an average tumor volume >900 mm3
after 12 days (Figure 9B). In contrast, the tumorꢀbearing mice treated with compound
5l had an average tumor volume of < 400 mm3 and had an inhibition rate of 38.1%
(Figure 9C). The dose of reference compound 7 was 7.5 mg/Kg; this treatment
obtained an antiꢀtumor effect with an inhibition rate of 29.2%, which was slightly
lower than that of compound 5l. The positive control group was treated once two days
with doxorubicin at a dose of 1 mg/kg for 12 days and showed an effect similar to that
of compound 5l with an inhibition rate of 37.6%.
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To determine whether the expression of cꢀMyc was consistently affected within
tumors by treatment with compound 5l and 7, we assessed the cꢀMyc (brown area in
Figure 9D) in tumor tissue using immunohistochemistry (IHC). The experimental
group exhibited significantly decreased expression relative to the negative control
group. The results indicated cꢀMyc downꢀregulation by compound 5l and 7 might
contribute to tumor inhibition in vivo.
Figure 9. Compound 5l and 7, doxorubicin, and saline were administered by i.p.
injection to athymic nude mice with human tumor xenografts established using A549
lung cancer cells. The mice were injected i.p. once a day for 12 days. The control
group was injected with 150 µL of saline. The positive control group (DOX) received
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doxorubicin by i.p. injection at a dose of 1 mg/Kg, once two days for 12 days.
Compound 5l and 7 were similarly administered to mice, once a day, at a dose of 6.25
mg/Kg, and 7.5 mg/Kg, respectively. The body weight (A) and tumor volume (B)
were recorded every two days, and the tumor weight was evaluated when the
treatment ended (C). The inhibition rate was calculated using (tumor weight in
drugꢀtreatment group) / (tumor weight in control group) and indicated on the column.
(D) The expression of cꢀMyc protein was identified by IHC, and pictures were taken
at 400 × magnification. Images of three independent samples in each group were
selected and presented for to give an overall illustration of the results.
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Binding Mode Exploration by Using Molecular Modeling. Molecular
docking was performed to provide a possible binding mode of compound 5h and 5l
with the cꢀmyc Gꢀquadruplex. The ligands were docked to the threeꢀdimensional
structures of cꢀmyc Gꢀquadruplex (PDB code: 2L7V, authors will release the atomic
coordinates and experimental data upon article publication) determined by solution
state NMR spectroscopy as mentioned in the introduction. The docking results of 5a,
5h and 5l to cꢀmyc Gꢀquadruplex with highest scores were shown in Figure 10A. The
docking results of 5h showed that the benzofuroquinoline scaffold could effectively
stack on the external 5’ꢀterminal Gꢀquartet, and the triazole containing side chain
were properly oriented and directed into the DNA grooves and fill the groove space,
which helped lock the ligand onto the cꢀmyc Gꢀquadruplex. Specifically, the triazole
moiety interact with the loop through Hꢀbond. The positively charged terminal group
was well accommodated in the groove towards the negatively charged phosphate
diester backbone, and formed electrostatic interactions and Hꢀbonds. In addition, by
comparing with the compound 5a and 5h, compounds with longer side chain gain
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better score (ꢀ9.137) than that with shorter side chain (ꢀ7.696), again, indicating that
longer side chains might have better interactions with the groove.
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Result of 5l demonstrated a similar binding mode to the cꢀmyc Gꢀquadruplex.
However, the orientation of the side chain was slightly different from the above two
compounds. The triazole moiety seemed to possess a πꢀπ stacking interaction with one
of the guanines in the Gꢀquartet. These results further reinforced our design idea that
the triazole might interact with the Gꢀquadruplex.
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Figure 10. Docking results of derivatives 5a (A), 5h (B), and 5l (C) onto the cꢀmyc
Gꢀquadruplex (top views onto the 5’ of the cꢀmyc Gꢀquadruplex and side views, PDB
code: 2L7V, authors will release the atomic coordinates and experimental data upon
article publication).
Conclusions
A series of derivatives with the incorporation of 1,4ꢀtriazole moiety by using
click chemistry have been designed, synthesized and evaluated as effective and
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selective cꢀmyc Gꢀquadruplex binding ligands. Results of FRET, SPR and MST
experiments suggested that positive charged terminal substituents played a crucial role
in the stabilization ability, binding affinity and the selectivity of the derivatives. It is
noteworthy that the introduction of 1,4ꢀtriazole moiety lead to positive impact on the
binding affinity of the ligands. In addition, derivatives with longer side chains (n=3)
possessed better activity to the cꢀmyc Gꢀquadruplex. In line with the in vitro assays,
subsequent bioassays demonstrated that the selected derivatives 5a, 5h, and 5l
specifically downꢀregulated cꢀmyc gene transcription and expression in Raji cells,
presumably through the stabilization of cꢀmyc Gꢀquadruplex structure. Results from
the flow cytometric assays suggested that 5a, 5h, and 5l could arrest Raji cell cycle in
G0/G1. In addition, RTCA assays indicated that these derivatives could remarkably
inhibited A549 cell proliferation, without influencing normal primary cultured mouse
mesangial cells. Altogether, the reported results proved that modification by using
click chemistry is an efficient approach for obtaining potent and selective cꢀmyc
promoter Gꢀquadruplex binding ligands.
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To be noted, the activities of these new derivatives actually were similar with
that of the reference compound 7. Good transcriptional inhibition activity but poor
selectivity in compound 7 indicated that this compound might have alternative targets
in vivo. On the other hand, we have to emphasize one thing that the improved
selectivity in the present study refers to the binding selectivity of TꢀBFQs on cꢀmyc
quadruplex against that on duplex DNA. The specificity of these compounds to a
certain Gꢀquadruplex (such as cꢀmyc) versus other quadruplex genes is still need to
further improvement.
ꢀ
Experimental section
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