Radiosensitizing effect of novel phenylpyrimidine derivatives on human lung cancer cells via cell cycle perturbation

Article abstract of DOI:10.1124/jpet.119.257717

Radiotherapy is one of the most common treatments for cancer, but radioresistance and injury to normal tissue are considered major obstacles to successful radiotherapy. Thus, there is an urgent need to develop radiosensitizers to improve the therapeutic o

Full text of DOI:10.1124/jpet.119.257717

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Title page
Radiosensitizing effect of novel phenylpyrimidine derivatives on human lung cancer
cells via cell cycle perturbation
Seung-Youn Jung, Ky-Youb Nam, Jeong-In Park, Kyung-Hee Song, Jiyeon Ahn, Jong Kuk Park,
Hong-Duck Um, Sang-Gu Hwang, Sang Un Choi and Jie-Young Song*
Division of Radiation Biomedical Research, Korea Institute of Radiological & Medical Sciences,
Seoul 01812, Republic of Korea. (S.-Y. J., J.-I. P., K.-H. S., J. A., J. K. P., H.-D. U., S.-G. H., J.-Y. S.)
Research Center, Pharos I&BT Co., Ltd., Anyang 14059, Republic of Korea. (K.-Y. N.)
Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul
0
3760, Republic of Korea. (K.-H. S.)
Bio & Drug Discovery Division, Korea research Institute of Chemical Technology, Daejeon 34114,
Republic of Korea. (S. U. C.)
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Running Title page
Running Title: Radiosensitizing effect of novel phenylpyrimidine compound
*Address correspondence to: Jie-Young Song, Division of Radiation Biomedical Research, Korea
Institute of Radiological & Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul 01812, Republic of
Korea. Tel.: +82 29701308; Fax: +82 29702402; E-mail address: immu@kirams.re.kr
Number of text pages: 26
Number of tables: 3
Number of figures: 4
Number of references: 45
Number of words in the Abstract: 224
Number of words in the Introduction: 387
Number of words in the Discussion: 1397
Non-standard Abbreviations:
CDK, Cyclin-dependent kinase; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; PARP, poly (ADP-ribose) polymerase; PBS, phosphate-buffered saline;
PI, propidium iodide; PPA, N-phenylpyrimidin-2-amine; RT, radiotherapy; γH2AX, gamma H2A
histone family member X
Recommended section assignment:
Chemotherapy, Antibiotics, and Gene Therapy
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Abstract
Radiotherapy is one of the most common treatments for cancer, but radioresistance and injury to
normal tissue are considered major obstacles to successful radiotherapy. Thus, there is an urgent need
to develop radiosensitizers to improve the therapeutic outcomes of radiotherapy in cancer patients.
Our previous efforts to identify novel radiosensitizers, using high-throughput screening targeting p53
and Nrf2 revealed a promising N-phenylpyrimidin-2-amine (PPA) lead compound; 17 derivatives of
this lead compound were examined in the present study. PPA5, 13, 14, 15, and 17 inhibited cell
viability by more than 50% with a marked increase in the proportion of cells arrested at the G2/M
phase of cell cycle. Among these compounds, PPA15 markedly increased the sub-G1 cell population
and increased the levels of cyclin B1 and the phosphorylation levels of cyclin-dependent kinases 1
(
CDK1). Combined treatment with radiation and PPA14 or PPA15 significantly decreased clonogenic
survival. An in vitro kinase assay revealed that PPA15 inhibited multiple CDKs involved in cell cycle
regulation. Compared with drug or radiation treatment alone, combined treatment with PPA15 and
radiation resulted in the suppression of A549 tumor growth in mice by 59.5% and 52.7%, respectively.
Treatment with PPA15 alone directly inhibited tumor growth by 25.7%. These findings suggest that
the novel pan CDK inhibitor, PPA15, may be a promising treatment to improve the effectiveness of
radiotherapy for the treatment of cancer.
Significance Statement (<120 words)
Several inhibitors of CDK have been successfully evaluated in combination with other
chemotherapeutics in clinical trials, but negative side effects have partially restricted their clinical use.
In this study, we identified a novel pan-CDK inhibitor to increase radiosensitivity, and we hope this
work will encourage the development of promising small-molecule radiosensitizers.
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Introduction
Radiotherapy (RT) is used to treat up to 50% of cancer patients, making it the most common
therapeutic strategy for cancers treatment (Chi et al., 2018). However, a considerable number of
patients undergoing radiotherapy develop radioresistance and experience recurrence, with a
consequent deterioration of the prognosis and a decreased survival rate. Recent advances in molecular
and cellular biology have revealed that several diverse factors play a role in the acquisition of
radioresistance in human cancers: the number and intrinsic radiosensitivity of cancer stem cells, tumor
hypoxia and reoxygenation during treatment, repopulation between radiotherapy fractions and
redistribution of surviving cells, and DNA damage repair (Petersen et al., 2001; Yaromina et al., 2011;
Krause et al., 2011; Baumann et al., 2016; Sharma et al., 2016). Therefore, the development of
effective radiation response modifiers that target the above-described relevant pathways is extremely
important for increasing the efficacy of radiotherapy (Harrington et al., 2011).
Cyclin-dependent kinases (CDKs) are a family of serine-threonine kinases that play a critical role
in cell cycle regulation (CDK1, 2, 4, and 6), transcriptional regulation (CDK7, 8, and 9), and neuronal
function (CDK5) (Asghar et al., 2015). Aberrations in CDKs, affecting cell cycle control and their
cyclin partners, have been observed in various cancers, usually characterized by dysregulated cell
division (Malumbres and Barbacid, 2001). Thus, CDKs have long been regarded as attractive targets
for the development of pharmacological inhibitors for cancer treatment. Many early, first-generation,
non-selective CDK inhibitors showed toxicity and poor efficacy; however, the recent development of
multi-target CDK inhibitors and more selective targeting of CDK4/6 inhibitors has led to the approval
of palbociclib for the treatment of breast cancer (O’Leary et al., 2016; Sherr et al., 2016).
In our attempts to identify novel radiosensitizers, we performed cell-based screening using a
luciferase-reporter assay system targeting tumor protein 53 (p53; TP53), nuclear factor (erythroid-
derived 2)-like 2 (Nrf2; NFE2L2), and transforming growth factor beta (TGF-β) (Lim et al., 2012;
Kang et al., 2015; Lee et al., 2012; Jung et al., 2015). A chemical library of 14,600 compounds was
screened, and we identified a hit compound containing an aniline-pyrimidine scaffold, as shown in
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Table 1. These chemotypes are known to be promiscuous kinase inhibitors and have been heavily
patented. Therefore, in this study, we synthesized several derivatives of the hit compound and
determined their biological activities and underlying mechanisms.
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Materials and methods
General methods for chemistry
All reactions involving air-sensitive reagents were performed in an inert atmosphere (N or Ar)
2
using syringe-septum cap techniques. All glassware was oven-dried prior to use. Mass spectra were
1
recorded on a JMS-700 MStation (EI mode; JEOL Ltd., Tokyo, Japan). The H NMR spectra were
recorded on an Avance-500 (Bruker Daltonics Inc. Billerica, MA, USA). Chemical shifts are
expressed as δ values relative to trimethylsilyl as the internal standard, J values are expressed in Hz,
and spectra were recorded in CDCl or hexadeuterodimethyl sulfoxide (DMSO-d ; Sigma-Aldrich, St.
3
6
Louis, MO, USA). Multiplicities of signals are indicated by the following symbol: s (singlet), d
doublet), dd (double doublet), t (triplet), m (multiplet) and brs (broad singlet). All reagents used were
(
acquired from Sigma-Aldrich and used without further purification unless otherwise stated. The N-
phenylpyrimidin-2-amine (PPA) compounds PPA1–PPA8 were purchased from ChemBridge (San
Diego, CA, USA).
4-(4-fluorophenyl)-N-(4-nitrophenyl)pyrimidin-2-amine (PPA9)
1
H NMR (DMSO-d , 400 MHz): δ = 10.5 (1H, s), 8.68 (1H, d, J = 4.0 Hz), 8.52 (1H, s), 8.23-8.32
6
+
(
3H, m), 8.10 (2H, d, J = 8.0 Hz), 7.62 (1H, d, J = 8.0 Hz), 7.42 (2H, t, J = 8.0 Hz). Mass (FAB ): m/z
+
3
11.1 (MH ). Yellow solid.
4-(4-fluorophenyl)-N,6-diphenylpyrimidin-2-amine (PPA10)
1
H NMR (DMSO-d , 400 MHz): δ = 9.75 (1H, s), 8.38-8.42 (2H, m), 8.32-8.34 (2H, m), 8.00 (1H,
6
s), 7.91 (2H, d, J = 8.0 Hz), 7.57-7.59 (3H, m), 7.42 (2H, t, J = 10.0 Hz), 7.35 (2H, t, J = 8.0 Hz), 6.99
+
+
(
1H, t, J = 8.0 Hz). Mass (FAB ): m/z 342.1 (MH ). Yellow solid.
N-(4-((4-(4-Fluorophenyl)pyrimidin-2-yl)amino)phenyl)acetamide (PPA11)
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1
H NMR (CDCl , 400 MHz): δ = 8.35-8.40 (3H, m), 7.54 (2H, d, J = 8.0 Hz), 7.37 (2H, d, J = 12.0
3
Hz), 7.18 (1H, brs), 7.13 (2H, t, J = 8.0 Hz), 6.75 (1H, brs), 6.53 (1H, d, J = 8.0 Hz), 2.20 (3H, s).
+
+
Mass (FAB ): m/z 323.1 (MH ). Yellow solid.
4-(4-fluorophenyl)-N-(4-(4-methylpiperazin-1-yl)phenyl)pyrimidin-2-amine (PPA12)
1
H NMR (CDCl ,400 MHz): δ = 8.35-8.41 (2H, m), 8.30 (1H, d, J = 4.0 Hz), 7.24-7.28 (2H, m),
3
7
.13 (2H, t, J = 8.0 Hz), 6.97 (2H, d, J = 8.0 Hz), 6.68 (1H, brs), 6.45 (1H, d, J = 4.0 Hz), 3.23 (4H, t,
+
+
J = 6.0 Hz), 2.60 (4H, d, J = 6.0 Hz), 2.37 (3H, s). Mass (FAB ): m/z 364.2 (MH ). Yellow solid.
4-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)-3-methoxy-N-methyl-benzamide (PPA13)
1
H NMR (CDCl , 400 MHz): δ = 8.35-8.41 (2H, m), 8.30 (1H, d, J = 4.0 Hz), 7.24-7.28 (2H, m),
3
7
.13 (2H, t, J = 8.0 Hz), 6.97 (2H, d, J = 8.0 Hz), 6.68 (1H, brs), 6.45 (1H, d, J = 4.0 Hz), 3.23 (4H, t,
+
+
J = 6.0 Hz), 2.60 (4H, d, J = 6.0 Hz), 2.37 (3H, s). Mass (FAB ): m/z 353.1 (MH ). Yellow solid.
4-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)benzenesulfonamide (PPA14)
1
H NMR (DMSO-d , 400 MHz): δ = 10.1 (1H, s), 8.62 (1H, d, J = 4.0 Hz), 8.26 (2H, dd, J = 10.0,
6
4
1
.0 Hz), 7.99 (2H, d, J = 8.0 Hz), 7.77 (2H, d, J = 8.0 Hz), 7.52 (1H, d, J = 4.0 Hz), 7.41 (2H, t, J =
+
+
0.0 Hz), 7.19 (2H, s). Mass (FAB ): m/z 345.1 (MH ). Bright yellow solid.
4-((4-(2-chlorophenyl)pyrimidin-2-yl)amino)benzenesulfonamide (PPA15)
1
H NMR (DMSO-d , 400 MHz): δ = 10.2 (1H, s), 8.66 (1H, d, J = 8.0 Hz), 7.96 (2H, d, J = 8.0 Hz),
6
7
.72 (2H, d, J = 8.0 Hz), 7.62-7.69 (2H, m), 7.52-7.56 (2H, m), 7.20 (1H, d, J = 4.0 Hz), 7.28 (2H, s).
+
+
Mass (FAB ): m/z 361.0 (MH ). Yellow solid.
4-(2-chlorophenyl)-N-(3-nitrophenyl)pyrimidin-2-amine (PPA16)
1
H NMR (DMSO-d , 400 MHz): δ = 10.3 (1H, s), 8.95 (1H, s), 8.69 (1H, d, J = 4.0 Hz), 8.08-8.15
6
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(
1H, m), 7.79-7.81 (1H, m), 7.70-7.73 (1H, m), 7.63-7.66 (1H, m), 7.52-7.59 (3H, m), 7.24 (1H, d, J =
+
+
4
.0 Hz). Mass (FAB ): m/z 327.1 (MH ). Green solid.
4-((4-(2-chlorophenyl)pyrimidin-2-yl)amino)-N-methylbenzamide (PPA17)
1
H NMR (DMSO-d , 400 MHz): δ = 10.0 (1H, s), 8.64 (1H, d, J = 8.0 Hz), 8.21-8.26 (1H, m), 7.87
6
(
2H, d, J = 12.0 Hz), 7.76 (2H, d, J = 8.0 Hz), 7.62-7.68 (2H, m), 7.51-7.54 (2H, m), 7.16 (1H, d, J =
+
+
4
.0 Hz), 2.75 (3H, d, J = 4.0 Hz). Mass (FAB ): m/z 339.1 (MH ). White solid.
Cell culture and treatment
Human lung carcinoma A549 and H1299 cells, prostate carcinoma DU 145 cells, ovarian
carcinoma SKOV3 cells, glioblastoma U-87 MG cells, and breast cancer MDA-MB-231 cells were
purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were
grown in RPMI 1640 or Dulbecco’s modified Eagle’s medium (Welgene, Daegu, Korea)
supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin
(
Invitrogen, Carlsbad, CA, USA). Cells were maintained at 37°C in a humidified atmosphere
containing 5% CO . All compounds were dissolved in DMSO and the final concentration of DMSO
2
did not exceed 0.1% (v/v). All controls were treated with DMSO only.
Cell viability assay
Cell viability was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(
MTT; Sigma-Aldrich) colorimetric growth assay. Briefly, cells were seeded in 96-well plates at a
density of 1,000 cells/well and treated with the indicated compounds for 72 h. All tested substances
were dissolved in DMSO and diluted to the indicated final test concentrations. MTT (5 mg/ml) was
added to the cells, and they were further incubated for 4 h at 37°C. The supernatant was removed, 100
μl DMSO was added, and then the absorbance was measured at 540 nm using a microplate reader
(
Multiskan EX; Thermo Fisher Scientific, Waltham, MA, USA). All experiments were performed in
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triplicate.
Apoptosis assay
Apoptosis was evaluated by measuring the proportion of annexin-positive cells. Cells were labeled
with allophycocyanin-conjugated annexin V and propidium iodide (PI) in binding buffer (BD
Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions and analyzed using a
FACSCalibur™ Flow Cytometer (BD Biosciences). For each sample, at least 10,000 events/sample
were acquired. The percentage of annexin V-APC-positive cells was analyzed with FlowJo software V
7
.2.5 (Tree Star Inc., Ashland, OR, USA).
Cell cycle analysis
2
A549 cells were seeded in 6-well plates at a density of 10,000 cells/cm and treated under the
indicated conditions. Cells were harvested and fixed in 75% ethanol for 1 h. They were then washed
with phosphate-buffered saline (PBS) and resuspended in PBS buffer containing 100 μg/ml RNase A
(
Sigma-Aldrich) and 50 μg/ml PI, followed by incubation at 37 °C for 30 min. A minimum of 10,000
events/sample were analyzed by flow cytometry to determine the DNA content. The percentage of
singlet cells in each cell cycle stage (sub-G1, G0/G1, S, G2/M, or aneuploid) was analyzed with
FlowJo software V 7.2.5.
Clonogenic assay
Cell survival after irradiation was determined using a clonogenic assay (Dunne et al., 2003). Briefly,
cells were seeded on 60-mm tissue culture dishes at various densities, treated with the indicated
1
37
compounds at 1 μM for 24 h, and then exposed to 3 or 3.5 Gy radiation with a Cs gamma irradiator
(
BioBeam 8000; Gamma-Service Medical GmbH, Leipzig, Germany). After 10-14 days, colonies
were fixed and stained with 1.0% methylene blue in absolute methanol solution for 10 min. Colonies
larger than 0.1 mm diameter were scored as surviving colonies. The experiment was performed in
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triplicate for each cell line.
Kinase assays
The activity of each compound, at a concentration of 10 μM and with an ATP concentration of 10
μM, was screened against a protein kinase panel of 106 human protein kinases provided by the
Eurofins’ KinaseProfiler™ Service (Eurofins Pharma Discovery Services, Dundee, UK). The half-
maximal inhibitory concentration (IC ) values were calculated from 10-point dose-response curves
50
provided by the Eurofins IC Profiler™ Service.
50
Western blot analysis
Total proteins from each cell lines was extracted in TNN buffer (50 mM Tris-Cl, pH 7.4; 1% NP-40;
50 mM NaCl, and 1 mM EDTA) supplemented with protease inhibitors (1 mM
1
phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 mM Na VO ) and
3
4
quantified using the Bradford method. Protein samples (15 μg) were separated by SDS-PAGE and
transferred to a nitrocellulose membrane. After blocking the non-specific antibody binding sites, the
membrane was incubated overnight at 4 °C with specific antibodies against p-cdc25C (S216) (63F9;
Cell Signaling Technology, Billerica, MA, USA), cdc25C (5H9; Cell Signaling), p-CDK2 (T160)
(
(
Cell Signaling Technology, Billerica, MA, USA), CDK2 (78B2; Cell Signaling), p-cdc2 (p-CDK1)
Y15) (10A11; Cell Signaling), cdc2 (PHO1; Cell Signaling), p-Histone H3 (p-HH3) (S10) (D2C8;
Cell Signaling), Histone H3 (HH3) (Cell Signaling), Cyclin A2 (BF683; Cell Signaling), Cyclin B1
D5C10; Cell Signaling), p53 (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p21
(
Waf/Cip1 (12D1; Cell Signaling), gamma H2A histone family member X (γH2AX; Santa Cruz
Biotechnology), poly (ADP-ribose) polymerase (PARP) (46D11; Cell Signaling), and β-actin (Sigma-
Aldrich). After incubation with peroxidase-conjugated secondary antibodies at 37°C for 1 h, the
protein bands were visualized using enhanced chemiluminescence reagent (GE Healthcare
Biosciences, Pittsburgh, PA, USA) and detected using the Amersham Imager 680 (GE Healthcare
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Biosciences). The relative levels of phosphorylated protein expression were normalized by to their
correspondent total protein and the relative levels of other protein expression were normalized to β-
actin.
Tumor xenograft mouse models
Female 6-week-old athymic nude (nu/nu) mice from Orient Bio Inc. (Seongnam, Korea) were
housed under specific pathogen-free conditions in microisolator cages with laboratory chow and water
6
ad libitum. A549 cell xenografts were established by implanting 1 × 10 cultured cells subcutaneously
6
0
into the thigh of the right hind leg of the mice. The tumor site was locally irradiated with 5 Gy Co γ-
3
radiation (2 Gy/min) when the tumor volumes reached 100–150 mm . PPA5 (10 mg/kg) or PPA15 (2
mg/kg) was administered intraperitoneally 3 h before irradiation. The tumor was measured along two
axes (L, longest axis; W, shortest axis) with Vernier calipers two or three times per week after
2
3
irradiation. Tumor volume was calculated as (L × W )/2 (mm ). All animal experiments were
approved by the Institutional Animal Care and Use Committees (IACUC) of the Korea Institute of
Radiological & Medical Sciences (KIRAMS 2018-0031).
Statistical analysis
Statistical analyses were performed using GraphPad software version 5 (GraphPad, La Jolla, CA,
USA). All data are expressed as the mean ± standard deviation (SD), except for the xenograft assay,
where data are expressed as the mean ± standard error (SE). Significant differences between groups
were determined using analysis of variance (ANOVA) and Bonferroni post-hoc comparisons. A value
of P<0.05 was considered statistically significant.
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Results
Chemistry
The structures of the PPA derivatives are shown in Table 1. PPA1–PPA8 were purchased from
ChemBridge, and the new PPA derivatives (PPA9–PPA17) were synthesized according to the methods
outlined in Scheme 1.
Effects of PPA derivatives on cell viability
The MTT assay was used to evaluate the in vitro cell viability after incubation with each compound at
a fixed concentration of 10 μM in A549 human lung cancer cells. Compared to the DMSO control,
compounds PPA4, PPA5, and PPA13–PPA17 displayed > 50% growth inhibition after 72 h (Fig. 1A).
To investigate the inhibitory effect of these compounds on cell growth, asynchronously growing A549
cells were exposed to each compound for 72 h and cell cycle analysis was performed (Supplemental
Table 1). As shown in Fig. 1B, PPA5, and PPA13–PPA17 significantly increased the population of
cells in G2/M. In addition, the population of aneuploidy cells with an abnormal number of
chromosomes (> 4n) (Orr et al., 2015; Pantazi et al., 2014) was also increased following treatment
with these five compounds, except PPA5. PPA14 induced the most evident arrest at the G2/M phase,
with 72.4% of the cell population in G2/M; only 9.0% of cells treated with PPA14 were in the G0/G1
phase compared to 78.4% of cells in the DMSO control. In addition, PPA15 significantly increased
the proportion of cells in the sub-G1 phase, an indicator of cell death, from 1.5% in control cells to 9.8%
(
Fig. 1C).
Radiosensitizing effect of PPA derivatives
Among the tested PPA derivatives, we selected six compounds (PPA5, and PPA13–PPA17) for further
investigation based on the above findings. Since highly proliferative cancer cells are more susceptible
to chemotherapy or radiotherapy during G2/M arrest (Guha, 2012), we aimed to determine whether
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the selected PPA compounds would increase the sensitivity of cancer cells to radiation. Consistent
with many earlier findings, irradiation alone increased the proportion of cells in the G2/M phase from
15.2% in the DMSO control to 41.1% (Supplemental Table 2). PPA5, PPA16, and PPA17 treatment in
conjunction with irradiation additively increased the population of cells in G2/M arrest after 48 h,
whereas treatment with PPA13–PPA15 alone caused a large population of cells to remain in the G2/M
phase. Given the results of Fig. 1C, G2/M arrest induced by each PPA5, PPA16, and PPA17 was
maintained for 72 h, while PPA13–PPA15-induced G2/M arrest occurred early, and released slowly.
Interestingly, an increase in the sub-G1 cell population was observed only with a combination of
PPA15 and irradiation, suggesting that PPA15 may be the most effective radiosensitizer compound
(
Fig. 2A and Supplemental Table 2). To further confirm the radiosensitizing effect of the selected
compounds, we measured clonogenic survival, which is the most reliable and classical method to
determine radiosensitivity (Buch et al., 2012). As shown in Fig. 2B and 2C, treatment with PPA13,
PPA14, and PPA15 alone increased the cytotoxicity of the cells. Moreover, the radiation-induced
clonogenic survival of A549 human lung carcinoma cells showed a greater decrease following
treatment with PPA14 or PPA15, suggesting that both these compounds possess radiosensitizing
activity.
PPA derivatives induce G2/M arrest
To further investigate the molecular mechanism underlying the induction of G2/M arrest by the
selected PPA derivatives, we measured the levels of proteins involved in G2/M phase progression in
p53 wild-type A549 and p53-null H1299 cells. PPA15 and PPA16 treatment increased the levels of
phosphorylated cdc25c (S216) in A549 cells, which is a suppressed state of cdc25c phosphatase
function and thus prevents cell progression from G2 to mitosis. In addition, increased levels of active
CDK2 (T160), inhibitory CDK1 (Y15), cyclin A2, and cyclin B1 expression by PPA14 and PPA 15 in
both cell lines indicated that cells remained primarily in the G2 phase. In particular, the treatment of
PPA14, PPA15, and PPA17 significantly decreased expression of phosphorylated histone-H3,
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indicating that the cells did not enter the mitotic phase or already exited the mitotic phase. The levels
of p53 in A549 cells were increased by some PPAs, while p21 expression was highly increased in
H1299 cells by PPA14 and PPA15 treatment. These results indicate that PPA15 effectively increased
the proportion of cells in the G2/M phase by modifying the activity of the cyclin/CDK complex in a
p53 independent manner.
Anti-tumor effect of PPA
To further evaluate the radiosensitivity of PPA15, we measured apoptotic cell death in the presence or
absence of p53 function. As shown in Fig. 4A, combined treatment with PPA15 and irradiation
significantly increased the population of annexin-positive cells in a p53 independent manner. It is
well-known that p53 and p21 are key regulators of cell cycle arrest and apoptosis, and that both are
involved in G1 as well as G2/M arrest after irradiation, thereby sensitizing tumor cells to radiation
(
Iliakis et al., 2003; Jung et al., 2017). As expected, irradiation markedly increased the protein levels
of p53 and p21 in p53 wild-type A549 cells, whereas PPA15 did not significantly alter p53/p21 levels,
but rather increased the levels of PARP1 cleavage and γH2AX expression. In p53-null H1299 cells,
p21 levels were slightly increased in response to both irradiation and PPA15, either alone or in
combination. In addition, PARP1 cleavages and γH2AX levels were also increased in the same
treatment (Fig. 4B). These data suggest that PPA15 may effectively increase radiation-induced cell
death via cell cycle perturbation with insufficient DNA damage repair.
Based on these results, the weakest (PPA5) and most active (PPA15) compounds were selected for
the evaluation of anti-tumor effects in an in vivo xenograft mouse model. Mice were administered
with 10 mg/kg PPA5 or 2 mg/kg PPA15 intraperitoneally 3 h prior to local irradiation. The body
weights of the mice during the whole experiment did not differ significantly between all groups.
Compared to the control, PPA5 alone, radiation alone, and combined PPA5 and irradiation treatment
inhibited tumor growth by 14.7%, 36.5%, and 70.9%, respectively (Fig. 4C). PPA15 treatment at 20%
that for PPA5 resulted in 25.7% tumor inhibition compared to control levels. PPA15 in combination
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with irradiation inhibited tumor growth more effectively than PPA15 alone or radiation alone, but a
synergistic effect was not observed. Despite the insignificant effect on radiosensitivity by PPA15 in
the animal study, the clonogenic survival of several irradiated cancer cells, including DU 145
prostates, SKOV3 cervical, U-87 glioma, and MDA-MB-231 breast cancer cells, was significantly
decreased by PPA15 at 1 μM (Fig. 4E, Supplemental Fig. 1), suggesting the usefulness of PPA15 as
an adjuvant for radiotherapy, with broad activity across multiple tumor types.
To identify the target molecule of PPA15, we performed an in vitro kinase assay. In contrast to the
non-specificity of PPA5, PPA15 showed highly selective inhibition of CDK/cyclin complexes (Table
2
). PPA15 majorly inhibited the kinase activity of p25/CDK5, cyclin E/CDK2, cyclin E/CDK3, and
cyclin A/CDK2 (IC , 9, 12, 18, and 19 nmol/L, respectively) and was less active against cyclin
50
B/CDK1 or cyclin D/CDK4 by at least several-fold (Table 3).
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Discussion
Although RT is a crucial and cost-effective strategy for the clinical treatment of cancer (Sharma et
al., 2016), its curative effect is not totally reliable. The need to improve the therapeutic outcomes of
radiotherapy gives rise to an urgent requirement for the discovery and clinical application of new
radiosensitizer drugs. The results of the present study indicate that PPA15 shows great potential as an
effective radiosensitizer, via cell cycle perturbation, for use in human cancer treatment.
The cell cycle is inextricably linked to the cellular response to radiation. Radiation-induced single
or double strand DNA breaks trigger multiple signaling pathways, including DNA damage responses
and the activation of cell cycle checkpoints (Johnson and Shapiro, 2010). In addition, cells in the
G2/M phase of the cell cycle are approximately three-times more radiosensitive than cells in the S
phase (Terasima and Tolmach, 1963). Furthermore, many cancer cells have mutations or deletions in
the p53 gene or other p53 signaling pathway defects that impair its role in controlling G1 checkpoint,
which leads to a far greater reliance on G2 checkpoints than that observed in normal cells (Kawabe,
2
004). Therefore, novel compounds could exert radiosensitizing activity by increasing the proportion
of tumor cells in a more radiosensitive cell cycle phase (Pauwels et al., 2010). The p53 tumor
suppressor protein plays a key role in numerous signaling pathways, including those involved in DNA
repair, cell cycle progression, redox systems, inflammation, senescence, apoptosis, and metabolism
(
Fei and El-Deiry, 2003; Levine and Oren, 2009). The PPA moiety was identified by performing cell-
based high-throughput screening to identify novel small molecules that increase radiosensitivity via
p53 modulation (Kang et al., 2015). Among the 17 derivatives of PPA investigated, 6 were selected
based on their ability to inhibit cell viability and induce G2/M arrest.
The combined treatment of the selected compounds with irradiation more strongly induced G2/M
arrest than each compound alone, except for PPA15, which stimulated the release of cells from arrest
and reentry into the cell cycle. Clonogenic survival analysis of the selected compounds demonstrated
that PPA15 was the most effective radiosensitizer. Since distinctive G2/M arrest was observed in
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response to PPA compounds, we examined their effect on the expression of cell cycle-related proteins
in two cell lines with different p53 status. The treatment-induced decrease in cyclin A/CDK2 and
increase in cyclin B/CDK2 levels allowed cells to enter the G2 phase. Concomitantly, inhibitory
cdc25c and CDK1 levels were increased, suggesting that cells were arrested in M phase without p53
dependency. In addition, the rapid release from G2/M arrest by PPA15 and irradiation treatment may
have induced cell death, as evidenced by the increase in the apoptotic cell population, PARP1
cleavage and γH2AX levels. Finally, the administration of a single dose of PPA15 (2 mg/kg)
significantly inhibited tumor growth in mice. However, the in vivo radiosensitizing effect of PPA15
was weakly observed in this study. Because the effect of candidate compounds in vivo is highly
dependent on experimental conditions, optimal PPA15 regimens, including dose, routes of
administration, and schedule of the combined irradiation will likely vary for different types of tumors
and will have to be determined accordingly. In addition, further pharmacokinetic/pharmacodynamic
analysis of PPA15 is required to obtain maximum efficacy.
According to the kinase inhibitory profile of PPA15, most CDK/cyclin complexes were inhibited by
PPA15. Thus, PPA15 is considered as a pan-CDK inhibitor (IC values for CDK1, CDK2, CDK3,
5
0
CDK5, and CDK9 were in the 10–100 nM range). The underlying functions of CDK3 and CDK5 are
currently unclear. CDK3 is intrinsically important for the entry of cells into the S phase via activation
of transcription factors belonging to the E2F family, but its role can be readily compensated by other
CDKs (Ye et al., 2001). CDK5 was largely considered a neuronal kinase that protected the nervous
system from damage. However, it was recently demonstrated that CDK5 has functions similar to those
of CDK4 and CDK2 in driving cell cycle progression from G1 to S in medullary thyroid cancer
models (Pozo et al., 2013). CDK9 is involved in basal transcriptional regulation via the
phosphorylation of RNA polymerase II and plays a crucial role in cell growth, proliferation, and
differentiation (Romano and Giordano, 2008).
CDKs are highly validated targets for cancer therapeutics because they play a critical role in the
regulation of cell cycle checkpoints and transcription. CDKs are major drug targets, and many CDK
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inhibitors have been described in the literature. However, because of the functional redundancy of
CDKs/cyclins and their critical roles in normal cellular growth, pharmaceutical strategies to develop
multiple-targeting or broad-spectrum CDK inhibitors remains limited, and are being pursued in
parallel (Satyanarayana and Kaldis, 2009). Redundant CDKs can easily compensate for the loss of
another’s function in cancer cells, which provides an acceptable therapeutic margin via differences of
their activity in normal cells versus cancer cells (Syn et al., 2018). Therefore, it would be anticipated
that simultaneously targeting a range of CDKs is more likely to be therapeutically effective than the
selective inhibition of specific CDKs. To date, optimized pan-CDK inhibitors or multi-target CDK
inhibitors have been extensively investigated, and some clinical benefits have been demonstrated.
However, numerous CDK inhibitors have shown disappointing results in clinical trials, possibly
owing to an insufficient understanding of their mechanism of action, the absence of biomarkers, and
the resulting inappropriate patient selection (Whittaker et al., 2017). Elucidation of the critical targets
of these multi-target inhibitors could allow us to better determine the most effective strategy for
deploying these agents in a clinical setting (Garrett and Collins, 2011).
Several studies have demonstrated that CDK2, CDK4, and CDK6 are not essential for cell cycle
regulation in normal cells (Kozar and Sicinski, 2005; Dean et al., 2010; Sawai et al., 2012). However,
CDK1 is known to be required for mammalian cell proliferation, and appears to be a key determinant
in stimulating the onset of mitosis (Santamaría et al., 2007). Therefore, inhibitors that target CDK1
frequently lack selectivity for cancer cells over normal tissues, and cause problematic effects such as
bone marrow suppression, as well as other adverse effects commonly associated with traditional
chemotherapy, such as nausea, vomiting, and diarrhea (Asghar et al., 2015). For example, the use of
Dinaciclib (MK-7965), which inhibits CDK1, 2, 5, and 9, has been attempted in patients with
leukopenia and thrombocytopenia (Kumar et al., 2015; Mitri et al., 2015). The clinical development of
the CDK1, 2, and 9 inhibitor AZD5438 was discontinued partially because of its low tolerability and
high variability in patients (Boss et al., 2010; Prevo et al., 2018). Targeted individual CDK inhibitors
could protect normal cells against some cancers, but might have therapeutic value against restricted
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tumors showing the expression and activation of theses specific CDKs. Nevertheless, selective
CDK4/6 inhibitors (Palbociclib, Ribociclib, Abemaciclib) have been licensed for the treatment of
hormone receptor–positive, retinoblastoma–positive breast cancer in combination with antihormonal
agents (Hamilton and Infante, 2016). Based on new findings that non-cell cycle-related CDKs are
associated with cancer in diverse ways, selective inhibitors of CDKs 5, 7, 8, 9, and 12 have been
developed. Moreover, alternative approaches to discovering allosteric sites, protein-protein
interactions, and peptidomimetic mechanisms are also underway to overcoming the selectivity
problem for ATP-competitive agents (Heptinstall et al., 2018).
The narrow therapeutic window of CDK-targeted inhibitors remains a huge obstacle to effective
treatment; nevertheless, certain drugs with an in correct range of CDK selectivity as well as the
combination of CDK1 inhibitors with other targeted therapies continue to be developed (Whittaker et
al., 2017; Xia et al., 2014; Wu et al., 2018). Many studies have suggested that CDK inhibitors often
do not lead to tumor shrinkage, especially in solid tumors, but rather result in greater cytotoxic effects
when used in combination with other chemotherapeutics or radiotherapy (Guha, 2012). Consistent
with the above findings, PPA15 alone directly decreased the tumor volume in A549 xenograft mice,
suggesting that it is a promising agent for cancer treatment, either alone or as part of a combination
therapy. DNA-damaging chemotherapeutic agents combined with radiotherapy is common treatment
strategy, but many concerns still needed to be addressed, including optimizing scheduling, appropriate
target selection, improved preclinical models, and biomarker-based patient selection.
In conclusion, PPA15 treatment induced cell cycle arrest at the G2/M phase and enhanced the
radiosensitivity of cancer cells via the inhibition of CDK1, CDK2, CDK3, and CDK5. Further studies
are needed to establish the optimal sequence and schedule of PPA15 administration in combination
with radiotherapy.
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Authorship Contributions
SY Jung and KY Nam contributed equally to this work.
Conducted experiments: SY Jung, KH Song, and JI Park.
Performed data analysis: SY Jung, KH Song, and JI Park.
Performed drug design and synthesized compounds: KY Nam.
Interpreted the data: J Ahn, HD Um, JK Park, and SG Hwang.
Wrote or contributed to the writing of the manuscript: SY Jung, KY Nam, SU Choi, and JY Song.
Supervised the research design and laboratory activities: JY Song.
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Footnotes
Financial support: This work was supported by the National Research Foundation of Korea [Grant
No. NRF-2017M2A2A7A01019214] and the Korea Institute of Radiological and Medical Sciences,
funded by Ministry of Science and ICT, Republic of Korea [Grant No. 50531-2018].
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Figure Legends
Fig 1. N-phenylpyrimidin-2-amine (PPA) derivatives can induce G2/M arrest. (A) Cell viability
of A549 cells at 72 h after treatment with 10 μM PPAs. Data are shown as the means ± SD of three
independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. (B) A549 cells were treated with
1
0 μM PPAs for 72 h and then subjected to cell cycle analysis. Data represent the mean of each cell
cycle population from three independent experiments. (C) Histograms of selected PPA compounds
based on one representative result of (B). M1: sub-G1 phase; M2: G0/G1 phase; M3: S phase; M4:
G2/M phase, M5: aneuploid cells.
Fig 2. Selected N-phenylpyrimidin-2-amine (PPA) compounds increase radiosensitivity. (A) Cells
were treated with the indicated PPAs (10 μM) for 24 h prior to a 5 Gy dose of γ-irradiation and
subjected to cell cycle analysis after further incubation for 24 h. One representative result from three
independent experiments is shown. M1: sub-G1 phase; M2: G0/G1 phase; M3: S phase; M4: G2/M
phase, M5: aneuploid cells. (B) A549 cells were treated with 1 μM PPAs for 24 h and then exposed to
a 3.5 Gy dose of irradiation. Cells were stained with methylene blue 10 days after irradiation, and
representative staining images are shown. (C) Surviving fractions of the cells from (B), as an indicator
of radiosensitivity. Data are shown as the mean ± SD of the normalized values for each drug alone
treatment group. *P < 0.05 and **P < 0.01.
Fig 3. N-phenylpyrimidin-2-amine (PPA) compounds modulate cell cycle-related protein
expression. (A) A549 and (B) H1299 cells were treated with the indicated PPA at 10 μM for 48 h. The
levels of the indicated proteins were determined with western blotting and quantified by densitometry
using Image J software. One representative result of three independent experiments is shown. The
intensities of phosphorylated proteins were normalized by the level of their correspondent total
proteins and the intensities of other proteins were normalized by the level of β-actin. Values are given
as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.
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Fig 4. N-Phenylpyrimidine-2-amine (PPA) derivative PPA15 promotes radiosensitivity in vitro
and in vivo. A549 and H1299 cells were treated with 10 μM PPA15 for 24 h, following treatment with
or without a 5 Gy dose of γ-radiation. (A) After 24 h, quantitative analysis of apoptosis was conducted
with the annexin V/PI assay using flow cytometry. Data are shown as mean ± SD of annexin-positive
cells from three independent experiments. (B) The levels of the apoptosis-related proteins were
determined by western blotting. One representative image from three independent experiments is
shown. Values are expressed as the mean ± SD from three independent experiments. (C) A549 (1 ×
6
1
0 ) cells were injected subcutaneously into the thigh hind leg of nude mice (n = 4 mice/group). One
week after tumor cell injection, either PPA5 (10 mg/kg) or PPA15 (2 mg/kg) was administered
intraperitoneally 24 h prior to local γ-radiation at a dose of 5 Gy. Data are shown as the mean ± SE of
tumor volume. The red and blue markers represent the statistical significance of PPA15 and PPA5,
respectively. (D) Cells from several tumor types were treated with 1 μM PPA15 for 24 h and exposed
to a 3 Gy dose of γ-radiation. Surviving clonal cells were visualized at 10–14 days after irradiation.
One representative result from three independent experiments is shown. *P < 0.05, **P < 0.01, ***P
<
0.001 vs. control; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. IR; ##P < 0.01, ###P < 0.001 vs. drug
alone.
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Table 1. Structure of N-phenylpyrimidin-2-amine analogs
3
R
2
4
5
R
R
NH
N
R
R
N
1
6
R
derivatives R1
R2
-
R3
R4
-
R5
R6
IUPAC Name
-(4-fluoruphenyl)-N-phenylpyrimidin-
4
PPA1
-
-
2-amine (ChemBridge, 9227565)
N-(4-methoxyphenyl)-4-
PPA2
PPA3
-OCH3
-
-
-
-
-
-
-
phenylpyrimidin-2-amine (ChemBridge,
223502)
9
N-phenyl-4-(pyridine-2-yl)pyrimidin-2-
amine (ChemBridge, 9201661)
6-chloro-N-(4-nitophenyl)-4-
PPA4
PPA5
PPA6
-NO2
-NO2
-
-
-
phenylquinazolin-2-amine
(
ChemBridge, 5808624)
4
-(4-fluorophenyl)-N-(4-nitrophenyl)-6-
phenylpyrimidin-2-amine (ChemBridge,
469711)
-
-
5
N-(4-((4-methyl-6-(phenylamino)
pyrimidin-2-yl)amino)phenyl)
acetamide
-NH-
CO-
CH3
-CH3
(
ChemDiv, G869-0095)
(
3-((6-chloro-4-phenylqyinazolin-2-
yl)amino)phenyl)((morpholino)
PPA7
PPA8
-
methanone (ChemBridge, 5960675)
Ethyl 4-((4-(4-fluotophenyl)-6-
phenylpyrimidin-2-yl)amino) benzoate
-
COO-
C H5
2
-
-
-
-
-
-
(
ChemBridge, 5475712)
4-(4-fluorophenyl)-N-(4-
PPA9
-NO2
-
nitrophenyl)pyrimidin-2-amine
4-(4-fluorophenyl)-N,6-
PPA10
-
diphenylpyrimidin-2-amine
2
9

JPET Fast Forward. Published on June 28, 2019 as DOI: 10.1124/jpet.119.257717
This article has not been copyedited and formatted. The final version may differ from this version.
JPET #257717
-
NH-
CO-
CH3
N-(4-((4-(4-Fluorophenyl)pyrimidin-2-
yl)amino)phenyl)acetamide
PPA11
PPA12
PPA13
-
-
-
-
-
-
4
-(4-fluorophenyl)-N-(4-(4-
methylpiperazin-1-yl)phenyl) pyrimidin-
-amine
-
-
2
-
CO-
NH-
CH3
4-((4-(4-fluorophenyl)pyrimidin-2-
yl)amino)-3-methoxy-N-methyl -
benzamide
-OCH3
4
-((4-(4-fluorophenyl)pyrimidin-2-
yl)amino)benzenesulfonamide
PPA14
-
-
-
4
-((4-(2-chlorophenyl)pyrimidin-2-
yl)amino)benzenesulfonamide
PPA15
PPA16
PPA17
-
-NO2
-
-
-
-
-
-
-
4-(2-chlorophenyl)-N-(3-
-
nitrophenyl)pyrimidin-2-amine
-
CO-
NH-
CH3
4-((4-(2-chlorophenyl)pyrimidin-2-
yl)amino)-N-methylbenzamide
3
0