European Journal of Medicinal Chemistry 207 (2020) 112704

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Design, synthesis and anti-cancer activity of pyrrole-imidazole

polyamides through target-downregulation of c-kit gene expression
Mi Zhang a, b, 1, Jing Liang a, b, 1, Shi-Kun Jiang a, b, 1, Ling Xu a, b, Yan-Ling Wu c, **,
Annoor Awadasseid a, b, Xiao-Yin Zhao a, b, Xu-Qiong Xiong a, b, Hiroshi Sugiyama d, ***,
Wen Zhang a, b, *
a
Lab of Chemical Biology and Molecular Drug Design, College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310014, China
b
Institute of Drug Development & Chemical Biology, Zhejiang University of Technology, Hangzhou, 310014, China
c
Lab of Molecular Immunology, Virus Inspection Department, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, 310051, China
d
Lab of Chemical Biology, Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Pyrrole-imidazole polyamide (PIP) can specifically bind in the B-DNA minor groove that has been used in
Received 30 April 2020 several biological applications, such as anti-cancer activity, gene expression and translation control, and
Received in revised form visualization of complex genomic areas. c-kit is a family member of the Tyrosine Kinase (RTK) type III
12 July 2020
receptor and plays a vital role in tumor growth, proliferation, differentiation, and cell apoptosis; however,
Accepted 28 July 2020
Available online 5 August 2020
its mutations and overexpression induce tumor dysfunction. Here, we designed and synthesized five
matched PIPs that can recognize and bind to the DNA sequence in the oncogene c-kit promoter region,
and evaluated their anti-cancer activity. The RTCA assay findings revealed that the PIPs would prevent
Keywords:
Pyrrole-imidazole polyamide
the proliferation of cancer cells A549 and SGC-7901. EMSA assay showed that the PIPs were actively
c-Kit gene interacting with the c-kit gene target DNA. RT-PCR and Western blot assays have an effect on expression
Anti-cancer activity levels of the c-kit gene in the presence of PIPs. Flow cytometry and wound-healing assays showed that
Apoptosis PIPs 4, 5 would cause apoptosis of cancer cells and inhibit the migration of cells, respectively. Overall
Drug findings indicate that PIP 5 has a relatively significant intracellular and extracellular impact on the target,
contributing to migration and proliferation reduction, and cancer cell apoptosis. In addition, PIP has a
certain ability to evolve into c-kit gene-targeting drugs.
© 2020 Elsevier Masson SAS. All rights reserved.

1. Introduction proliferation, cell growth, migration, and differentiation capacity,

The proto-oncogene c-kit, found on human chromosome 4 q11-
q12 [1], encoded an orphan transmembrane glycoprotein called
CD117, which belongs to the family members of the type III tyrosine
kinase receptor (RTK) [2]. CD117 has played a significant role as a
tyrosine kinase receptor for stem cell factor in tumor growth,
proliferation, differentiation, and apoptosis of cells [3e6]. Over-
expression and mutation of c-kit have significantly promoted cell
* Corresponding author. Lab of Chemical Biology and Molecular Drug Design,
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou,
310014, China.
** Corresponding author.
*** Corresponding author.
E-mail addresses: ylwu@cdc.zj.cn (Y.-L. Wu), hs@kuchem.kyoto-u.ac.jp
(H. Sugiyama), wzhang63@zjut.edu.cn (W. Zhang).
1
These authors contributed equally to this work.
contributing to the creation of numerous malignant tumors [7],
which involve Mast cell, acute myeloid leukemia (AML), gastroin-
testinal stromal tumors (GISTs), lung cancer, colorectal carcinoma,
melanoma, and testicular cancer [6,8e15]. Consequently, sup-
pressing the c-kit signaling pathway was found to be an important
therapeutic strategy for anticancer [16e18]. Imatinib, which in-
hibits the function of c-kit kinase, has had a strong inhibitory
impact on tumors induced by c-kit mutation or overexpression,
such as GIST. However, as imatinib was used for long-term care of
patients as a first-generation tyrosine kinase inhibitor, it would
cause c-kit gene mutation, which created acquired resistance in
later patient therapy, resulting in lower efficacy of Imatinib [19]. C-
kit inhibitors of Sunitinib and Regorafenib [20,21], as second-line
therapy drugs, solved the problem of clinical treatment failure of
imatinib. But due to the emergence of secondary c-kit mutations,
Sunitinib and Regorafenib, as same as Imatinib, still had limited

https://doi.org/10.1016/j.ejmech.2020.112704
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
2 M. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112704
therapeutic effect on patients with advanced cancer who expressed
excessive wild-type c-kit or mutant c-kit [22]. Hence, innovative
gene-targeted medicines will offer a new solution for the cure of
cancer induced by the c-kit malfunction.
Polyamides were a kind of small synthetic molecules similar to
the Distamycin A and could specifically bind in the minor-groove of
B-DNA [23]. Pyrrole-imidazole polyamides (PIPs) specifically
recognize the DNA base pairs (Py binding to C, Im binding to G, b (b-
alanine) binding to A or T) [24]. Thus, according to ‘recognition
rules’, PIPs could be designed to bind to different sequences of the
target genes, and their affinity was more than or equivalent to
natural DNA binding protein, which inhibited the signaling of
oncogenic transcription factors and reduced their binding at spe-
cific loci [25,26]. For example, PIPs, targeted the human trans-
forming growth factor TGF-b1 gene promoter, could inhibit the
transcription and expression of TGF-b1 in human cells and then
inhibited the formation of marmoset’s hypertrophic scar [27].
What’s more, PIPs targeting the TGF-b1 gene promoter were ex-
pected to become a practical gene-drug for the treatment of hy-
pertrophic scars. In further research, it was found that polyamides
through target downregulating TGF-b1 expression can effectively
improve marmoset’s nephropathy [28], and can also effectively
improve diabetic nephropathy [29]. With continuous rational
design and optimization, the development of small molecule
polyamides would quickly be promoted as a new powerful gene-
regulator drug for treating cancer and other diseases [30e35].
The structure of PIPs affected their biological activity; thus, the
intelligent design of a polyamide structure will improve their
binding affinity and sequence selectivity to target DNAs [36]. In the
pre-design process of PIP, in order to obtain higher specificity and
lower off-target effect rate, a longer-chain PIP was required [30].
However, an extension of the PIPs to eight or more residues would
cause the structure to bend excessively and no longer match the
shape of the small grooves of B-DNA, resulting in the loss of
hydrogen bond strength, thereby weakening the binding affinity
and specificity [30]. Our previous studies showed that introduction
of (S)-b-hydroxyl-g-aminobutyric acid residue (Sgb-OH) into PIPs
could increase the specific recognition ability to T base; meanwhile
an Sgb-OH/b (b-alanine residue) pair may relax the rigid curvature of
polyamide, raise polyamide matching degree with the DNA minor
groove and enhance the affinity between both molecules [37,38].
Additionally, (S)-a-hydroxyl-g-aminobutyric acid residue (Sga-OH)
at a-position of the g-aminobutyric acid turn (g-turn) in hair-pin
polyamide can effectively identify T base [39].
Until now, we don’t find that small or macro-molecules drugs
targeting nucleic acids of c-kit gene are used in the clinic and also
any reported information that polyamides were designed to pre-
cisely recognize c-kit DNA sequences in B-DNA minor groove and
regulate c-kit gene expression. Thus, based on the above studies
and findings, five types of PIPs are designed, synthesized, and
applied to target-downregulate c-kit gene expression through
specific binding to the core promoter sequence, 50 -AGGGGAGGC-
GAGGAGGGGCGTGGCCGG-30 containing sp1 binding site [40],
thereby suppressing tumor cell growth and causing tumor cell
apoptosis. We attempt to explore promising polyamide drugs tar-
geting c-kit nucleic acids against cancers, filling the gap of clinically
qualified c-kit’s kinase drugs.
2. Materials and methods
2.1. Compounds
PIPs were designed, synthesized, and characterized in our lab-
oratory (Fig. 1A, Scheme S1). The sample was dissolved in dimethyl
sulfoxide (DMSO) with a concentration of 10 mM as a stocking
solution and stored at 20  C for biological experiments.
2.2. Electrophoretic mobility shift assay (EMSA)
The 27 nt double-stranded DNA (ds-DNA) within the human c-
kit gene promoter region, 50 -AGGGGAGGCGAGGAGGGGCGTGGC
CGG-3’/30 -TCCCCTCCGCTCCTCCCCGCACCGGCC-5’ (Fig. 1B), with
stated purities of 95%, was bought for further experiments from
Sangon (Shanghai, China). The 27 nt ds-DNA was mixed with
different concentrations of polyamides. The ratio of PIP to DNA is 0 :
1, 0.5 : 1, 1 : 1, 2 : 1, 4 : 1, 8 : 1 and 16 : 1. A mismatch polyamide
(Fig. 1A) was used as the reference. DNAs and PIPs complexes were
separated by electrophoresis on 16% polyacrylamide gels and
stained by SYBR Gold (Invitrogen, USA). After 30 min staining, A Gel
documentation system (BIORAD) was used in the analysis of the
result.
2.3. Real-time cell analysis (RTCA) assay
All human cancer cell lines, including four human gastric cancer
cell lines (BGC823, MKN45, SGC-7901, MGC803), two human liver
cancer cell lines (H7402, BEL7402), a human pancreatic cancer cell
line JF305, a human breast carcinoma cell line (435S), a human lung
cancer line (A549), a human colon cancer cell line (HT29), a human
cervical cancer cell line (HeLa), were obtained from the China
General Microbiological Culture Collection Center (CGMCC, Beijing,
China). GIST-T1 cells were obtained from YaJi Biological, Shanghai.
Experiments were conducted according to the report [41]. In brief,
the cells were cultured in RPMI 1640 or DMEM medium (Gibco)
containing 10% fetal bovine serum (FBS), 1% Hepes (Gibco) and 1%
streptomycin-penicillin (Invitrogen) at 37  C in a 5% CO2 incubator.
The cells pellet by 0.25% trypsin-EDTA (Gibco). The cell growth
curves were continuously monitored by the xCELLigence Real-Time
Cellular Analysis system (Roche, Germany). The cell index (CI)
indicating the cell vitality was measured with an incubation time of
cells in the presence/absence of PIPs. 6  103 cells were seeded per
microtiter well, after 24 h growth, treated with the PIPs in various
concentrations (0, 1, 5, 10, 20, and 40 mM). The impedance was
determined up to 48 h after seeding. The data from the RTCA sys-
tem was processed by GraphPad Prism 8.0.
2.4. Wound-healing assay
The effect of the PIPs on migration in A549 and SGC-7901 cell
lines was studied through the wound-healing assay according to
the literature [41,42]. Previous reports showed that these two cell
lines have a robust migration ability [43,44]. 20  104 cells were
seeded into each well (24 well plate) and grown to 80%. The wound
at the bottom of the well was created through the tip of the pipette.
The cells were then washed twice with PBS (Gibco) to remove non-
adherent cells and photographed through an inverted fluorescent
microscope. The cells were then treated with serum-free medium
encompassing different concentrations of PIPs (0, 0.5, 1, and 1.5 mM)
for 48 h. The cell migration was evaluated by measuring the dis-
tance that migrated from the wound edge. Data were analyzed by
Image J.
2.5. Morphological analysis
A549 and SGC-7901 cells were incubated at 2  105 cells/well in
12-well flasks. After 24 h, the original medium was discarded, and
then the medium containing PIP 4 or PIP 5 was added to the wells,
in which the concentration of PIP 4 and 5 were 7.5 mM in SGC-7901-
containing well, while for A549-containing well were 15 mM. After
36 h of co-cultivation with PIPs and cells, cell morphology was
 M. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112704 3

Fig. 1. Chemical structure of PIPs (A) and the binding mode (B) of PIPs to the DNA sequence in the c-kit gene promotor. Amongst PIPs, 1e5 are the matched structures of
polyamides, and 6 (Mis-polyamide) is a mismatched structure of a polyamide according to the recognition rule of polyamide. The DNA sequence (81 to 107) is selected as a target
sequence, 50 -AGGGGAGGCGAGGAGGGGCGT GGCCGG-3’/30 -TCCCC TCCGCTCCTCCCCGCACCGGCC-50 , because it contains a portion (83 to 107) of fragment essential for maximal
core promoter activity of the c-kit gene, which includes 5 binding sites for 5 PIPs, located 100 to 104 for PIPs 1 and 2, e100 to 105 for PIP 3, e91 to 101 for PIP 4 and e95
to 101 base pairs (bp) upstream for PIP 5, respectively, and an essential binding site underlined with a dot (84 to 93) of the SP1 protein promoting transcription of the c-kit
gene.

observed under a Microsystems microscope (Leica, Germany).
2.6. Flow cytometry
In a twelve-well plate, 105 cells of A549/SGC-7901 were plated
per well, and after 20 h, PIPs 4 and 5 were added at final concen-
trations of 0, 10, and 20 mM, and 0, 7.5 and 15 mM, respectively. After
36 h, with trypsin digestion without EDTA, cells were collected and
then adding Annexin V and PI staining (BD Biosciences). Finally,
cells were tested on the CytoFLEXS (BECKMAN COULTER).
Apoptosis analysis was performed using flowJo_V10 (BD
Biosciences).
2.7. RNA preparation and semi-quantitative RT-PCR
Total RNA was extracted from A549 and SGC-7901 cell lines using
the QIAamp RNA Blood Mini Kit (QIAGEN, 52304, Germany)
according to the manufacture instructions after treatment with PIPs
4 and 5 at different final concentrations of 0, 5, 10 and 15 mM, and 0,
2.5, 5 and 7.5 mM, respectively, for 36 h. The total RNA concentration
was measured using the Nanodrop 2000 (Thermo, USA) and checked
by 1.5% agarose gel electrophoresis with ethidium bromide (EB)
staining. 400 ng total RNA was used as the template for each reverse
transcriptase (RT)-mediated PCR (RT-PCR) reaction using the TaKaRa
One Step RNA PCR Kit (Takara, Dalian). The primer sets for the c-kit
gene were 50 CAAGGCTTCTCCAATTCTGC-30 and 50 - TGCAGTGGTC-
CACAGAAGAG-3’. b-actin was used as the internal control to
normalize all samples for potential variations in mRNA content. The
primer sets for b-actin were 5-AGCGGGAAATCGTGCGTGACA-30 and
50 -GTGGACTTGGGAGAGGACTGG-3’. The thermal cycling was started
with a 30 min reaction at 50  C to reverse transcript from the mRNA
to cDNA followed by 2 min reaction at 94  C to activate the Taq
polymerase. 30 cycles for c-kit gene and b-actin at 94  C for 30 s,
62  C for 30 s and 72  C for 60 s. The PCR product was stored at 4  C.
4 M. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112704
Fig. 2. Extracellular interaction of PIPs with the c-kit gene promoter sequence by
electrophoretic mobility shift assay (EMSA). Lane 1: SSC; lane 2: SSG; Lane 3: ds-
DNA; Lanes 4e9: PIP þ ds-DNA (The ratio of DNA to PIP is 1:0.5; 1:1; 1:2; 1:4; 1:8;
1:16); Lane 10: Mis-polyamide (6, Fig. 1A) þ ds-DNA (a Mismatched polyamide was
used as reference). SSC is a 27 nt C-rich single-stranded DNA as a 27 nt complementary
strand for G-rich DNA and SSG a 27 nt G-rich single-stranded DNA in double-stranded
DNA (Fig. 1B), both as references. All experiments were performed in a buffer (pH 7.4)
containing 10 mM TriseHCl, 10 mM KCl, 0.1 mM EDTA, and samples of 2 mM DNA
dissolved in the buffer in the presence and absence of compounds were incubated
overnight after annealing at 95  C. The reaction mixtures were resolved by electro-
phoresis (16% native polyacrylamide gel, 150 V, 3 h) and visualized using SYBR GOLD
staining. Pre-preparation for all samples were shown in the methods section.
2.8. Western blot assay
The expression level of c-kit protein in the tumor cells treated
with PIPs is measured by Western blot assay. In brief, SGC-7901 or
A549 was treated with PIPs 4 and 5 at different final concentrations
of 0, 10, and 15 mM, and 0, 5, and 7.5 mM, respectively, for 36 h, and
total protein was extracted with RIPA lysate (Beyotime, Shanghai).
The protein concentration was measured by BCA kit (Beyotime,
Shanghai). The target protein was separate from 30 mg total protein
by 10% SDS-PAGE and transferred it to the PVDF membrane. After
the PVDF membrane with target protein was blocked with 5% BSA
for 2 h, it was incubated overnight with an antibody of c-kit and b-
tubulin, at 4  C. The secondary antibody was applied for 2 h, and
finally, ECL luminescent reagent (Beyotime, Shanghai) was added to
the image in a gel imager.
2.9. Distribution of fluorescein-labeled polyamide in vitro
A suitable number of SGC-7901 cells were inoculated in a petri
dish and cultured for 24 h. Then 1.0 M fluorescein (Fluo)-labeled PIP
5 was added and incubated with the cells for 4 h. After the incu-
bation, the original culture medium of cells was replaced with fresh
culture medium, and cells continued to be cultured for 20 h, then,
viewed at  300 under live cell conditions. Subsequently, cells were
fixed with 4% paraformaldehyde for 10 min, and then nuclei were
stained with Hoechst33342 (Invitrogen) for 5 min. After dyeing, the
cells were washed with PBS, and finally, the fluorescence was
measured in a confocal laser scanning microscope (TCS SP5 II, Leica,
Germany).
2.10. Circular dichroism (CD)
A Jasco J-815 circular dichroism (CD) spectropolarimeter was
used for the CD assay [41]. The quartz cuvette with a path length of
5 mm was selected, and the spectral data was collected at a scan-
ning rate of 100 nm/min at a bandwidth of 5.0 nm. The PIPs were
gradually dropped into a 3 mM of 27 nt of ds-DNA solution at the
ratio of PIP to DNA (r), 0.0, 0.1, 0.2, 0.3. , 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,
2.0, 3.0 into the target ds-DNA in the buffer (pH 7.0) at room
temperature, and after each addition, the CD spectrum from 225 to
400 nm was recorded, and the average of three parallel experi-
ments was obtained. After the equilibrium was reached, the data
were acquired by scanning for 15 min.
2.11. Molecular docking
The combination of polyamide and DNA sequence of the c-kit
gene promoter region is demonstrated by Discover Studio 4.0 [38].
The module of Discover Studio 4.0 is used to construct PIPs 4 and 5
with the standard bond length and bond angle as default settings,
and construct c-kit ds-DNA (Fig. 1B)in macromolecule using the
standard B-DNA helix parameters. Based on the NMR structure of the
ImPyPy-g-PyPyPy-50 -d(CGCTAACAGGC)-3’/5’ -d(GCCTGTTAGCG)-30
complex model [45], PIPs 4 and 5 were each docked with the DNA
minor groove. The composite was thoroughly infiltrated in an
aqueous solution. The entire composite system was carried out en-
ergy minimization in the CHARMm force field. When the system was
optimized to RMS <0.001 kcal/mol Å, all restrictions between PIP and
c-kit dsDNA were removed. Under the same conditions, the energy of
the entire system was minimized to RMS <0.001 kcal/mol Å.
2.12. Statistical analysis
The statistical significance of experimental findings was
measured using t-tests or one-way ANOVA analysis by unpaired
Student. Three independent experiments presented data as
means ± standard deviations (SD). The difference (P < 0.05) was
found significant as contrasted with the control group.
3. Results
3.1. Design, synthesis and binding mode of polyamides to c-kit gene
sequence
The structures of the polyamides and the sequence of the c-kit
gene used in this study are shown in (Fig. 1). The oligomer of the c-
kit DNA was synthesized by Sangon Biotech (Shanghai). The 27 nt
 M. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112704 5

Fig. 3. (A) c-kit gene expression in 11 types of cell lines. Up: the gel image of c-kit expression in the cell lines, lane 1: H7402, lane 2: JF305, lane 3: BGC-823, lane 4: 435S, lane 5:
BEL7402, lane 6: MKN-45, lane 7: SGC-7901, lane 8: HELA, lane 9: A549, lane 10: MGC-803, lane 11: HT-29; down: a histogram of gel bands with one-to-one correspondence. (B) and
(C) Antiproliferative ability of PIPs 4 (B), 5 (C), and imatinib mesylate (D) as a positive control to four cell lines: Bel-7402, MKN-45, SGC-7901, A549 cells. The histogram is the cell
survival rate relative to the control group after 48 h of adding drugs. Using the control group with a survival rate of cells being 100% as the standard, the survival rate of tumor cells
after adding different concentrations of PIPs (1, 5, 10, 20, 40 mM) was calculated by RTCA data (Fig. S1). Survival: 1e100%  [AUC-48 (Control) e AUC-48 (polyamide)]/AUC-48
(Control). AUC-48 (Control): area under concentration-time curve 48 h after the addition of 1% DMSO solution in RPMI 1640. AUC-48 (polyamide): area under concentration-time
curve 48 h after addition of PIPs. The values are expressed as mean ± SD (triplicates).

Table 1
kit gene and an important binding site of the SP1 protein promoting
IC50 values of four types of cancer cell lines treated with PIPs 4, 5, and imatinib
mesylate as a positive control for 48 h by RTCA. The values are expressed as
transcription of the c-kit gene. It was also reported that the c-kit
mean ± SD (triplicates). gene sequence, 50 -GGCGAGGAGGGGCG TGGCCGGC-30 , can be used
as the optimal site intervened in regulating c-kit expression levels
Compound IC50 (mM)a
for intervention to regulate c-kit expression levels by the dynamic
Bel-7402 MKN-45 SGC7901 A549 interaction research [47]. Consequently, modifying the fragment’s
4 14.87 ± 0.41 19.92 ± 0.79 6.05 ± 0.23 7.93 ± 0.31 structural properties and/or occupying the SP1 binding region may
5 11.19 ± 0.28 7.41 ± 0.35 2.96 ± 0.08 5.97 ± 0.14 significantly reduce the promoter’s transcriptional activity [40],
imatinib mesylate 55.52 ± 2.65 12.18 ± 0.29 0.208 ± 0.047 1.87 ± 0.06
influencing the expression level of a c-kit proto-oncogene. For this
core sequence of the c-kit gene promoter, five PIPs and one mis-
a
IC50 value: Drug concentration producing 50% cancer cell death/proliferation
inhibition. matched PIP were designed, synthesized (Fig. 1A, Scheme S1), and
their interaction mode with target DNA sequence was shown
(Fig. 1B) according to the polyamide recognition rule.
double-stranded DNA (ds-DNA) sequence, 50 -AGGGGAGGCGAGGA
GGGGCGTGGCCGG-3’/30 -TCCCCTCCGCTCCTCCCCGCACCGGCC-50 , 3.2. Extracellular interaction investigation of PIPs with the target (s)
located 81 to 107 bases upstream of the transcription start site by electrophoretic mobility shift assay (EMSA)
(TSS), is selected as a target sequence for PIPs [3,40,46], because it
contains a portion (83 to 107) of fragment essential for maximal The polyamide can specifically recognize the nucleic bases and
core promoter activity of the c-kit gene, which contains 5 binding bind in the minor groove of B-DNA [48]. EMSA assay is a useful
sites for 5 PIPs at upstream of the initiation of transcription of the c- method for investigating the interaction of a small-molecule drug
6 M. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112704

Fig. 4. The effect of PIP 4 and PIP 5 on cell migration by the wound-healing assay on SGC-7901 (A) and A549 (B) cell lines. Data were shown as Mean ± SD (n ¼ 3). * *
*P < 0.001, significantly different from untreated control group.

Fig. 5. Morphological changes and apoptosis of SGC-7901 and A549 cells induced by PIPs. (A) Morphological changes induced by PIPs 4 and 5. (B) Cell apoptosis induced by PIP
5. Cells were treated with PIPs 4 and 5 (7.5 mM, 15 mM) for 36 h, cell morphology was observed under a Microsystems microscope, and apoptotic cells were analyzed by Annexin V-
FITC/PI flow cytometry. The histograms indicated the proportion of apoptosis from three separate experiments. Data are presented as the Mean ± SD (n ¼ 3). *P < 0.05, **P < 0.01,
significantly different compared with the untreated control group.
 M. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112704 7

Fig. 6. mRNA (A) and protein (B) expression levels of the c-kit gene in SGC-7901 and A549 cells treated with the PIP 5 for 36 h by RT-PCR analysis and Western blot,
respectively. A series of PIP 5 concentrations: 0, 2.5, 5, 10 mM, and 0, 5, 10 mM in A and B were used. b-actin and b-tubulin were used as internal references in A and B, respectively.
The histogram dates were derived from Western blot results by quantitative analysis of image J. (C) Distribution of fluorescein (Fluo)-labeled PIP in vitro. SGC-7901 cells were
incubated with Fluo-labeled polyamide for 24 h and examined by fluorescence microscopy under live cells condition and then fixed and viewed again. Nuclei were stained with
Hoechst 33342 (blue). Magnification,  300 in C. Data are presented as the Mean ± SD (n ¼ 3). **P < 0.01, ***P < 0.001, significantly different compared with the untreated control
group.

and DNA/protein and between nucleic acid and protein through the
change of bands on gel [49,50]. As shown in (Fig. 2), PIPs 1, 2 and 3
did not have observable effects on bands of the double-stranded
target DNA sequence of the c-kit gene promoter (Fig. 1B) at
experimental concentrations ranging from 0 to 32 mM, suggesting
that the interaction cannot be observed between 3 polyamides and
the target DNA by EMSA. While the PIP 4 gave rise to obvious
interaction with the target ds-DNA at the DNA/PIP concentration
ratio of 1: 8, as the second slow-moving band appeared. The second
slow-moving band was considered as species of PIP-DNA complex.
With increasing compound concentration, the second slow-moving
band gradually became more dark and obvious in a compound
concentration-dependent manner. Still, even at the highest con-
centration of 32 mM, the fast-moving band for the target ds-DNA
did not disappear.
It was further interesting that, relative to PIP 4, PIP 5 displayed
the very good binding capability to the target ds-DNA and even at
the 1:2 DNA/PIP concentration ratio, the faster-migrating band was
fully transformed to the slower-migrating one, indicating that PIP 5
had the best binding capability to the ds-DNA sequence in the c-kit
gene promoter region of the five matched PIPs examined. Although
at the highest concentration of 32 mM, the mismatch compound as
a reference did not give rise to any detectable interaction with the
target DNA. Taken together, these data demonstrate that designed
PIPs, especially 4, 5, could specifically recognize and bind to the ds-
DNA sequence of the c-kit gene. Thus, PIPs 4 and 5 were selected for
further research.
3.3. PIPs inhibited proliferation of cancer cells by real-time cell
analysis (RTCA) technique
The above extracellular assay had proved that PIPs 4 and 5 could
be strong target-interact with the c-kit DNA, in particular, PIP 5.
Owning to abnormal expression of the c-kit gene was observed in
several malignancy cells [6], to validate the effects of PIPs on other
tumor cells, we analyzed expression level of c-kit gene in 11 rep-
resentatives of tumors cell lines by RT-PCR, including 4 human
gastric cancer cell lines (BGC823, MKN45, SGC-7901, MGC803), 2
human liver cancer cell lines (H7402, BEL7402), a human pancreatic
cancer cell line JF305, a human breast carcinoma cell line (435S), a
human lung cancer line (A549), a human colon cancer cell line
(HT29), a human cervical cancer cell line (HeLa). It can be clearly
seen from (Fig. 3) that lanes 5, 6, 7, and 9 are brighter than other
lanes, and the histogram intuitively also showed the different
expression levels of the c-kit gene in 11 cell lines, of which c-kit
gene expression in SGC-7901, BEL7402, MKN45, A549 cell lines was
higher than that of other cell lines, in particular, highest in SGC-
7901 cells. JF305 and H7402 cell lines have low expression of the c-
kit gene. Therefore, we selected 4 types of cell lines BEL7402,
MKN45, SGC-7901, and A549 with the high c-kit expression for
subsequent experiments.
As an advanced exceptional technique and tool for evaluating
the interaction of drugs with their targets, The RTCA (Real-time cell
analysis) system has the advantage of full automation, high sensi-
tivity, and high accuracy and label-free detection. It can monitor
cell status in real-time. In the process of cytotoxicity screening, it
can directly reflect the status of cell proliferation and cell survival
by using the cell index in real-time [51]. Here, we obtained in detail
evaluation for inhibitory effect and apoptotic ability of PIPs and
imatinib mesylate as a positive control on BEL7402, MKN45, SGC-
7901, and A549 cells by RTCA assay. As shown in (Figs. S1AeD),
PIPs 1e5 exhibited different inhibitory activity for tested cancer
cells under experimental conditions. It was worth of our special
attention that, amongst five PIPs, PIP 4 and 5 possessed more
8 M. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112704
Fig. 7. The CD spectral properties of PIPs 4 (A) and 5 (B), of which the insets in A
and B are the change of CD with the r ([PIP4]/[DNA]) at 329.0 nm and the r ([PIP5]/
[DNA]) at 331.6 nm respectively. To specifically binding to the 27 nt target ds-DNA
sequence in the c-kit gene promoter (Fig. 1B), which is a portion (e83 to e107) of
fragment essential for maximal core promoter activity of the c-kit gene. A sample
of 3 mM ds-DNA (in double-strand) was dissolved in the buffer containing 10 mM
sodium cacodylate, 100 mM NaCl, 0.5% DMSO, incubated for 24 h after annealing at
95  C, centrifuged for degassing and then used at once for CD experiments. All CD
spectral experiments were performed by titration of the PIPs at the ratio of a com-
pound to DNA (r), 0.0, 0.1, 0.2, 0.3. , 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0 into the target
ds-DNA in the buffer (pH 7.0) at room temperature. The spectral data were collected at
a scanning rate of 100 nm/min at a bandwidth of 5.0 nm and the average of three
parallel experiments obtained. The PIPs were gradually dropped into a 3 mM target
DNA solution under defined conditions. After the equilibrium of each addition was
reached for 15 min, the CD spectrum from 225 to 400 nm was recorded. Dot curve:
DNA alone; dash line: polyamide alone, which was used at the concentration of 10 mM.
Solid arrows denote the increase of the polyamide concentration, and a dash one
makes isoelliptic points.
potent interaction effects with IC50 values of less than 20 mM on
tested cells, but PIP 2 and 3 on SGC7901 cells, in an almost dose-
dependent manner (Figs. S1, 3B, 3C, Table 1). In particular, PIP 5
presented most potent inhibitory effect and apoptotic ability on
SGC-7901 and A549 cells with IC50 values of 2.96 ± 0.08 and
5.97 ± 0.14 mM, respectively (Table 1). However, relative to imatinib
mesylate with IC50 values of 0.208 ± 0.047 and 1.87 ± 0.06 mM
(Figs. S1E and 3D, Table 1), PIP 5 also had low activity on both
cancer cells. This implies that PIPs actually ought to be more opti-
mized as extremely active drug candidates.
In order to confirm whether knockdown of c-kit gene affect
exmined cell growth, we chose small interfering RNA (siRNA) to
knock down the expression of c-kit gene in A549 cells, and further
confirmed siRNA effect. First of all, three siRNAs (si-c-kit-1, si-c-kit-
2 and si-c-kit-3) were used to examine their inhibition efficiency on
the expression of c-kit in A549 cells by western blot experiment. As
shown in Fig. S2, we found that si-c-kit-1 can significantly knock
down the expression of c-kit in A549. Thus, we chose si-c-kit-1 to
complete the next similar experiments. To verify whether poly-
amide can also exert anti-tumor effect on the classical cell models,
such as GIST-T1 cells, we chose PIP 5 to conduct this experiment,
which has shown good extracellular and intracellular effect. Results
in Figs. S3A and B confirmed that, compared with the control group,
PIP 5 can inhibit the expression of c-kit gene at both the tran-
scriptional and translational levels, in particular, give rise to strong
inhibition at the translational level even at 5 mM (Fig. S3B). Also, the
c-kit gene expression in GIST-T1 cells was greatly knocked down by
using siRNA, especially at 50 nM of si-c-kit-1 (Figs. S3C and D) and
the proliferation of GIST-T1 was inhibited in the presence of si-c-
kit-1 (Fig. S3E). Furthermore, we investigated the effect of PIP 5
on the proliferation of GIST-T1 cells. As seen in Fig. S3F, results by
RTCA revealed that PIP 5 displayed strong an antiproliferative effect
on human GIST-T1 cells under our experimental condition. There-
fore, it is confirmed that PIP 5 can also achieve good anti-tumor
effect through c-kit pathway in the classical cell model, such as
GIST-T1 cells.
Taken together, these findings showed that PIPs, especially 4
and 5, are capable of inhibiting the proliferation of tested cancer
cells besides GIST-T1 cells and promoting apoptosis as well as
supporting extracellular experimental results.
3.4. PIPs suppressed the mobility of human SGC-7901 and A549 by
wound healing assay
It was reported that more than 90% of cancer cell migration
plays an important role in cancer progression, and more than 90%
of the cancer patients are killed by metastases [52]. The wound-
healing assay is a simple method to detect cell motility. With
serum-free culture, the proliferation of cells may be neglected, so
the wound between cells will gradually become smaller due to cell
migration.
In our study, we examined the effect of PIPs 4 and 5 on A549 and
SGC-7901 cell migration by this method. As shown in (Fig. 4,
Fig. S4), cancer cell migration was influenced by the PIPs and in a
compound dependent manner. PIPs 4 and 5 displayed quite strong
inhibition effect with 80.77% and 85.70% of migration inhibition on
SGC-7901 cells, respectively, and 73.49% and 83.76% of that on
A549 cells, respectively, at the concentration of 1.5 mM, indicating
that PIP 5 had a better inhibitory effect. The motility of SGC-
7901 cells was more strongly inhibited than that of A549 cells at the
highest tested concentration. This result is in accord with that ob-
tained by RTCA assay.
3.5. PIPs induced morphological changes and apoptosis of SGC-
7901 and A549 cells by flow cytometry technique
To further investigate the anti-cancer activity of PIPs, we first
detected, under a microscope, the morphological modifications of
SGC-7901 and A549 cells exposed to 7.5 mM and 15 mM of PIPs 4 and
5, respectively. As shown in Fig. 5A, SGC7901 cells were detached
from the culture dish bottom and became round and formed
apoptotic bodies after being exposed to 7.5 mM of PIP 4 or PIP 5 for
36 h. They were noticeably smaller, and the interior appeared to be
fragmented, suggesting that the tumor cells underwent apoptosis.
Although A549 cells treated with 15 mM of PIP 4 or PIP 5 were partly
detached from the bottom of the culture dish, they clearly became
less, exhibited unusual morphological changes, and the membrane
 M. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112704 9

Fig. 8. A molecular simulation study of the recognition of PIP 5 with a b/Sgb-OH to the target DNA sequence of the c-kit gene. (A) A molecular binding model of the 1:1 complex
of PIP 5 to DNA (Fig. 1B) in the minor groove, 50 -AGGGGAGGCGAGGAGGGGCGTGGCCGG-3’/30 -TCCCCTCCGCTCCCCCCCGC ACCGGCC-50 obtained by energy minimization by the use of
NOESY-derived semi-quantitative distance constraints. The polyamide is shown as a ball model, with oxygen in red, hydrogen in gray, nitrogen in blue, and carbon in green. (B) A
close-up and schematic presentation of binding of PIP 5 with T/A base pair for partial intermolecular contacts between the b/Sgb-OH pairing moiety and the AT base pair with an
additional hydrogen bond between the b-OH of Sgb-OH and O2 of base T at the length of 2.21 Å, For DNA, the T base is denoted in red, A base in blue, sugar ring in yellow. (C) A close-
up and schematic presentation of binding of PIP 5 with T/A base pair for partial intermolecular contacts between the a-NHþ3 group in the g-turn at the g-turn position of polyamide
and the O2 of base T and The sugar ring oxygen atom O40 with additional hydrogen bonds at the length of 2.71 and 2.06 Å respectively. For DNA, the T base is denoted in red, A base
in blue, sugar ring in yellow. A dotted line denotes hydrogen bonding.

bulged outward, suggesting the same apoptotic process as
SGC7901 cells, albeit weakly affected them. Then, we employed
Flow cytometry (FCM) commonly used to detect the occurrence of
apoptosis [53] to investigate the apoptotic ability of PIPs to SGC-
7901 and A549 cells using PI/Annexin V FITC kit. After cancer
cells were treated with PIP 5 for 36 h, results showed that apoptosis
rates of SGC-7901 and A549 cells were dose-dependent and
reached 54.5% and 48.5% respectively at the concentration of 15 mM
(Fig. 5B), while treated with 20 mM of PIP 4, those of both cells were
53.3% and 47.9% respectively (Fig. S5). The data suggest that PIP 5
had a more obvious killing effect on SGC-7901 cells than PIP 4.
3.6. PIPs inhibited the expression level of c-kit gene and distribution
monitoring of PIP in vitro
Based on the extracellular and intracellular results by EMSA and
RTCA, wound healing, and FCM assays, we concluded that PIPs
could interact with a target DNA sequence in the promoter region of
the c-kit gene and induce the apoptosis and/or inhibit the prolif-
eration of tested cells. To clearly understand the molecular mech-
anism for PIPs leading to such intracellular results and to ascertain
whether the suppression of tumor cell growth was caused by
examined PIPs downregulating overexpression of the c-kit gene in
tested cancer cells. We subsequently investigated the expression
level of mRNA and protein of the c-kit gene in SGC7901 and
A549 cells in the presence or absence of the PIPs 4 and 5 at different
concentrations for 36 h through RT-PCR and Western blot,
respectively. As shown in Fig. 6A and B, PIP 5 specifically and dose-
dependently decreased the c-kit gene expression in both tested
cells, in particular, mRNA expression at 7.5 mM compared with non-
treated groups. While when the concentration of PIP 4 reached
15 mM, which was the maximum two-fold concentration of PIP 5,
PIP 4 inhibited the expression of the c-kit gene in both cancer cells
(Fig. S6). Collectively, these data showed that PIPs 4 and 5 owned a
strong ability to downregulate c-kit gene mRNA and protein
expression in SGC7901 and A549 cells, interestingly enough, PIP 5
had very strong inhibition of mRNA expression at 7.5 mM. This is one
of possible reasons leading to cytotoxicity through the down-
regulation of the c-kit gene by PIPs from the above intracellular
assays.
Next, we examined the ability of si-c-kit-1 to knock down c-kit
gene expression, causing the proliferative inhibition or apoptosis of
A549 cells. Results indicated that si-c-kit-1 inhibited the c-kit gene
expression at the transcriptional and translational levels (Figs. S7A
and B) and the proliferation of A549 cells (Fig. S7C), suggesting that
there is a definite relationship between the c-kit expression and the
10 M. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112704
proliferation of A549 cells. This hints that inhibiting the expression
of c-kit in non-small cell lung cancer may be an effective method
for the treatment of ineffective or recurrent c-kit positive tumors
[54].
In addition, in order to further ascertain that PIPs interact with
target DNA in the cell nucleus, the distribution of fluorescein (Fluo)-
labeled PIP 5 in SGC-7901 cells after 24 h incubation was monitored
by a fluorescence detector in (Fig. 6C). We clearly observed the
Fluo-labeled polyamide could be localized in nuclei and interact
with DNA like a nucleic acid dye, Hoechst 33342.
3.7. Investigation of binding ability and properties of PIPs 4 and 5 to
the target DNA sequence in the c-kit gene promoter by circular
dichroism (CD) assay
To in detail illuminate the binding properties of novel poly-
amides at an extracellular level to the target DNA sequence(s) in the
c-kit gene promoter (Fig. 1B), including binding strength, the ki-
netics of their appearance, their interaction modes and stoichi-
ometry of polyamide-DNA complexes and so on, Circular Dichroism
(CD) spectroscopy has been applied in our research, which is
regarded as a pioneering approach for understanding the interac-
tion of small molecules with nucleic acids [41,55,56]. Of six novel
PIPs including five matched polyamides 1e5 and one mismatched
polyamide 6, based on the previous experimental results, PIPs 4, 5,
and 6 as negative control were selected as representatives used in
this study (Fig. 1A).
As shown in Fig. 7A and B, CD spectra of the target ds-DNA alone
(dotted line) presented a robust positive band at 266.0 nm and a
relatively shallow negative band at 238.0 nm, which is character-
istic of a double-stranded DNA. In contrast, PIP 4 alone is optically
active and owns CD signals at 299.0 nm and 329.0 nm (dashed line,
Fig. 7A), PIP 5 alone is also optically active, but does quite small CD
signals between 297.0 nm and 332.0 nm (dashed line, Fig. 7B). More
importantly, in Fig. 7A and B, CD spectrum of the target ds-DNA, in
particular polyamide-induced CD (ICD), displayed the significant
difference in peak intensity, position, shape, and change, indicating
that different polyamide has different structural/electronic prop-
erties when bound to the target DNA. In Fig. 7A, with the pro-
gressive titration of PIP 4 in the target ds-DNA at the specified
concentration gradients, the positive peak amplitude of the ds-DNA
CD spectrum at 266.0 nm gradually increased up to saturation at a
ratio of 1:1 polyamide to DNA, and the negative bands at 238.0 nm
nearly did not shift. In contrast, in Fig. 7B, with gradual titration of
PIP 5, that of the ds-DNA CD at 266.0 nm almost no changed,
maximum positive peak at 266.0 nm blue-shifted to that at
261.5 nm and the negative bands at 238.0 nm progressively became
strong up to saturation at 1:1 ratio of polyamide to DNA with a
maximum ellipticity of 11.0. Further, with the increase of poly-
amide concentration, in Fig. 7A, positive shoulder bands ranging
from 278 nm to 311.0 nm gradually became more and more strong
up to saturation at 1:1 ratio. A negative ICD band between 311.2 nm
and 382.0 nm noticeably increased to a maximum negative peak
with an ellipticity of 9.25 at 329.0 nm. While in Fig. 7B, PIP 5
presented completely contrary cotton effects of ICD spectrum to PIP
4, and quite strong ICD bands, especially positive ICD bands,
ranging from 266.0 nm to 385.0 nm appeared, including a
maximum negative peak at 297.2 nm and a positive peak with a
maximum ellipticity of 26.80 at 331.6 nm. This suggests that there
exists a strong interaction between PIPs 4, 5, and the target ds-DNA
of the c-kit gene, respectively. The CD profiles have an isoelliptic
point at 311.2 nm in Fig. 7A and two isoelliptic points at 250.5 nm
and 208.1 nm in Fig. 7B, indicating that there are two ICD bands of
the positive and negative cotton effects. In addition, in the insets of
Fig. 7A and B, that the negative CD signals at 329.0 nm and the
positive ones at 331.6 nm, respectively, were plotted against
compoundeDNA ratio, both indicates stoichiometry of a 1:1
polyamide-DNA complex, presumably their interaction mode being
by binding of PIP 4 and 5 to the target ds-DNA in DNA minor groove
[38]. Likewise, the CD spectra for the binding ability of Mis-
polyamide 6 as a negative control to the target ds-DNA were ob-
tained (Fig. S8), showing that there was very weak interaction be-
tween both molecules.
To sum up, these results suggest that there exists a stronger and
more durable binding of PIPs 4 and 5 to the target ds-DNA in the c-
kit gene promoter at the ratio of 1:1, which is consistent with gel
electrophoresis results shown in Fig. 2 and supports the above
experimental results.
3.8. Molecular docking study of the interaction of PIPs 4 and 5 with
the c-kit gene DNA sequence in the minor groove
In order to clearly understand the binding mode and affinity of
PIPs to the matched target DNA sequence of the c-kit gene pro-
moter, we conducted a molecular simulation study on their inter-
action (Figs. 8 and S9). As seen in Fig. 8B and Fig. S9B, it can be
found that, as PIP 5 contains an Sgb-OH residue, the hydroxyl of Sgb-
OH can form a hydrogen bond with O2 of base T with shorter
bonding length of 2.21 Å compared with that of 2.87 Å for that
formed between PIP 4 and base T [37,38]. More importantly, there
were two additional hydrogen bonds formed between the a-NHþ 3
group in the g-turn of PIP 5 and the O2 of base T and the sugar ring
oxygen atom O40 with bonding lengths of 2.71 and 2.06 Å respec-
tively. Relative to PIP 4 (Fig. S9B), of which the same a-position is
linked to three bigger continuous N-methylimidazole (Im) residues
(ImImIm) (Fig. 1A) [39]. To quantitatively illuminate the binding
ability of PIPs, we calculated the binding energy of PIPs 4 and 5 to
the target DNA by the Discover Studio 4.0 (Table S1). This result
showed that PIP 5 possessed more binding stability to the target
DNA than PIP 4; this may be because there is a destabilization of big
steric hindrance of three continuous Im’s at the g-turn position of
PIP 4. In short, the molecular docking study indicated that PIPs 4
and 5 could better recognize and bind to the target DNA in B-DNA
minor grooves according to ‘recognition rule’. And PIP 5 exhibits
stronger binding ability than PIP 4 partly due to additional
hydrogen bonds, which is in agreement with that by the above
other assays.
4. Discussion
Cancer has always been a major disease that threatens the
health of human life, whereas the incidence of lung cancer and
stomach cancer maintain at a high level [57]. c-kit mutations (e.g.,
GIST) and overexpression (e.g., non-small cell lung cancer NSCLC)
not only cause dysfunction leading to tumors, but an aberrant
expression of c-kit also led to poor postoperative surgery [13].
Previous studies found that c-kit positive expression in non-small
cell lung cancer increased the mortality of patients [13]. At pre-
sent, clinical drugs targeting c-kit kinase generally become less
effective due to the appearance of c-kit secondary mutations.
Therefore, many drug-researchers are actively looking for new
ways to solve this problem [58]. Pyrrole-imidazole polyamide (PIP)
is a kind of promising small-molecule drugs for therapeutic gene
modulation [30]. What’s more Kurmis AA et al. reported that PIPs
were more effective than enzalutamide and bicalutamide in cell
culture, as well as active against enzalutamide-resistant prostate
cancer, demonstrating that polyamides can also make a difference
in drug resistance [59]. PIPs can exert biological activity against
normal or mutated mtDNA in specific ways [60]. Meanwhile, unlike
RNA drugs such as microRNA-664 [61], PIPs can be taken up by cells
 M. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112704
and easily enter the nucleus without the aid of vectors [28].
Thus, in this study, we designed five matched polyamides that
reverse-targeted c-kit. The EMSA experiments used to verify the
binding of compounds to the target sequence in vitro showed that
PIPs 4 and 5 could strongly bind to the target sequence. At a certain
concentration, PIPs 4 and 5 both inhibited the transcription and
translation of c-kit in A549 and SGC-7901 cells, cancer cell migra-
tion and induced apoptosis of SGC-7901 and A549 cells. It was
obvious that longer chain structures for PIPs 4 and 5 were better
than short-chain ones for PIPs 1e3 in relevant experiments, indi-
cating that appropriate long PIPs in the minor groove may give rise
to strong affinity and high specificity to B-DNA [62,63]. When
comparing PIP 4 with PIP 5, PIP 5 generally has an optimal effect at
extracellular and intracellular levels. The two owns very similar
structures; only one difference was that the PIP 4 contains an
additional three consecutive N-methylimidazole (Im) residues
(ImImIm) at the a-position of g-turn. It was reported that PIPs
containing more Im residues, especially three consecutive ImImIm
and more, are a bit difficult to permeate across cellular membranes
to reach their specific target DNAs [64], which is a key factor
affecting the intracellular activity of PIPs. Another reason is that PIP
5 possesses two additional hydrogen bonds formed between the a-
NHþ 3 group at its g-turn position and the base T and the sugar ring
compared with PIP 4 confirmed by molecular docking study (Figs. 8,
S6B, Table S2), leading to increase of binding ability of PIP 5 to
target ds-DNA. Also, it is not necessary to rule out an electrostatic
reaction between the positively charged amino group and the
negatively charged sugar/phosphate backbone. In addition, the
introduction of ImImIm residues at the g-turn position possibly
increases PIP 4 targeting ability. Meanwhile, it increases the rigidity
of the compound and steric clash with the DNA minor groove,
significantly weaken the affinity of PIP 4 to DNA.
In summary, a well-designed small molecule polyamide can
achieve therapeutic effects by inhibiting the c-kit expression, and
PIP 5 has a potential for further development as an anti-cancer drug
candidate or leading compound. To our knowledge, this is the first
time to report the polyamide targeting the c-kit gene.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
This work is supported by the National Natural Science Foun-
dation of China (No. 21877101) and the Zhejiang Leading Innova-
tion and Entrepreneurship Team (2018R01015).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112704.
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