1
PI-273, a substrate-competitive, specific small molecule inhibitor of PI4KIIα,
inhibits the growth of breast cancer cells Running title: The First PI4KIIα
Substrate-Competitive Specific Inhibitor
Jiangmei Li1, 7 #, Zhen Gao1, 6 #, Dan Zhao2, 6#, Lunfeng Zhang1,6, Xinhua Qiao1, 6, Yingying Zhao1,
7, Hong Ding2, Panpan Zhang 5,6,7, Junyan Lu2, Jia Liu7, Hualiang Jiang2, Cheng Luo2,4*, Chang
Chen1, 3, 6*
1National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules,
Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District,
Beijing, 100101, China
2Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, Shanghai, China
3Beijing Institute for Brain Disorders, No. 10 Xitoutiao, You An Men, Beijing, 100069, China
4The State Key Laboratory of Toxicology and Medical Countermeasures, Academy of Military
Medical Science, Beijing, China
5 Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China
6University of Chinese Academy of Sciences, 19 Yuquan Road, Shijingshan District, Beijing,
100049, China
7Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai,
China
#These authors contributed equally to this work.
Running title: The First PI4KIIα Substrate-competitive Specific Inhibitor
Key words: Phosphatidylinositol 4-kinase IIα; Substrate-competitive small-molecule inhibitor;
Virtual screening; Breast cancer
Grant Support: This research was supported by National Key R&D Program of China
(2017YFA0504000, 2016YFC0903100 to C. Chen), Personalized Medicines-Molecular
Signature-based Drug Discovery and Development, the Strategic Priority Research Program of
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the Chinese Academy of Sciences (XDA12020316 to C. Chen), the National Natural Sciences
Foundation of China (31500693 to C. Chen, 31101021 and 81472839 to J. Li., and 81430084,
81625022 and 21472208 to C. Luo), the ‘863’ National High-Technology Development
Program of China (0A200202D03 to C. Chen), National Key Scientific Instrument &
Equipment Development Program of China (2012YQ03026010 to C. Luo), NNCAS-2012-2 to
C. Chen, the Beijing Natural Science Foundation (7132156 to J. Li) and the Science and
Technology Commission of Shanghai Municipality (15431903100 to J. Li)
*Correspondence should be addressed to: Chang Chen, National Laboratory of
Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road,
Chaoyang District, Beijing 100101, PR China. E-mail: [email protected]. Tel:
86-10-64888406. or Cheng Luo, Drug Discovery and Design Center, State Key Laboratory of
Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555
Zuchongzhi Road, Pudong District, Shanghai, 201203, PR China. E-mail: [email protected].
Tel:86-21-50271399
Competing financial interests: The authors declare no competing financial interests.
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Abstract
While phosphatidylinositol 4-kinase PI4KIIα has been identified as a potential target for
antitumor therapy, the clinical applications of PI4KIIα are limited by a lack of specific
inhibitors. Here we report the first small-molecule inhibitor (SMI) of human PI4KIIα.
Docking-based and ligand-based virtual screening strategies were first employed to
identify promising hits, followed by two rounds of kinase activity inhibition validation.
2-(3-(4-Chlorobenzoyl)thioureido)-4-ethyl-5-methylthiophene-3-carboxamide (PI-273)
exhibited the greatest inhibitory effect on PI4KIIα kinase activity (IC50 = 0.47 μM) and
suppressed cell proliferation. Surface plasmon resonance (SPR) and thermal shift assays
indicated that PI-273 interacted directly with PI4KIIα. Kinetic analysis identified PI-273
as a reversible competitive inhibitor with respect to the substrate phosphatidylinositol
(PI), which contrasted with most other PI kinase inhibitors that bind the ATP binding
site. PI-273 reduced PI4P content, cell viability, and AKT signaling in wild-type MCF-7
cells but not in PI4KIIα knockout MCF-7 cells, indicating that PI-273 is highly selective
for PI4KIIα. Mutant analysis revealed a role of palmitoylation insertion in the
selectivity of PI-273 for PI4KIIα. Additionally, PI-273 treatment retarded cell
proliferation by blocking cells in G2/M, inducing cell apoptosis and suppressing
colony-forming ability. Importantly, PI-273 significantly inhibited MCF-7 cell-induced
breast tumor growth without toxicity. PI-273 is the first substrate-competitive,
subtype-specific inhibitor of PI4KIIα, the use of which will facilitate evaluations of
PI4KIIα as a cancer therapeutic target.
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Introduction
Phosphatidylinositol (PI) is an essential phospholipid that serves as a metabolic
precursor for both phosphoinositides and inositol (Ins) phosphates (1), and its related
phosphatidylinositol kinases (PIKs) play important roles in a number of human diseases,
including cancer (2-4), malaria (5), Alzheimer's disease (6-8) and diabetes (9). Among
the phosphatidylinositol kinases, PI4KIIα is targeted to the trans-Golgi network (TGN)
and endosomes. In mammalian cells, PI4KIIα is the dominant lipid kinase generating
PI4-phosphate (PI4P) (10), which is not only the precursor of the important regulatory
phosphoinositides PI(4,5)P2 and PI(3,4,5)P3 but also is an emerging regulatory molecule
in trans-Golgi (11) and endosomal trafficking (12,13), as well as phagocytosis (14).
PI4KIIα knockout mice develop late-onset neurological features that resemble human
hereditary spastic paraplegia (15). Recently, an increasing number of reports have
identified PI4KIIα as a potential target for breast cancer therapy (3,4,16-19). We
previously reported that increased PI4KIIα expression promotes tumor growth by
altering the HER2/PI3-kinase (PI3K)/ERK signaling cascades (4) and that dual
inhibition of EGFR and PI4KIIα represents a novel strategy to combat EGFR-dependent
tumors (17). PI4KIIα is also important for the Wnt (20,21) and Akt (3) signaling
pathways. However, in contrast to the extensive study on PI3Ks, pharmacological
manipulation and basic scientific research on PI4KIIα has been limited, largely owing to
a lack of specific inhibitors.
Numerous small-molecule inhibitors (SMIs) of PIKs have been identified via
screening in the past two decades, but most are ATP-competitive inhibitors of PI3Ks
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(22). Only
2-methyl-5-nitro-2-[(6-bromoimidazo[1,2-α]pyridin-3-yl)methylene]-1-methylhydrazid
e-benzenesulfonic acid (PIK-75)(23,24) and NCGC00012848-02 (NIH-12848) (25)
have been identified as substrate-competitive inhibitors. Isoform selectivity is
complicated by high conservation of the nucleotide-binding pocket amino acid
sequences of the different PIK isoforms, the site at which most SMIs bind. Because
isoform-selective inhibitors may reduce toxicity by decreasing off-target effects,
substrate-competitive inhibitors are urgently needed.
By combining crystal structure data, molecular dynamics (MD) simulations, and
biochemical and mutagenesis studies, we recently identified the putative PI-binding
pocket for PI4KIIα (26). The PI-binding pocket is lined by residues E157, Y159, R181,
L184, T307, D308 and L349 on the palmitoylation insertion and activation loop. In
addition to contributing to the PI-binding pocket, the palmitoylation insertion also
interacts with the membrane via an amphipathic helix, α4, and any perturbation that
weakens its membrane interaction significantly impairs its kinase activity. This insertion
does not occur in other PIKs, making it an ideal site for screening specific inhibitors. In
addition, a high-throughput and non-isotopic assay for PI4-kinase activity has been
developed (27), thereby enabling screens for PI4KIIα-specific inhibitors among large
compound libraries. Structure-based virtual screening (SBVS) has become a powerful
tool in the tool kit of medicinal chemists for rapidly enriching hits from large pools of
virtual compound libraries (28). In this study, we identified a PI substrate-competitive
SMI for PI4KIIα that notably inhibits tumor growth in a breast cancer model without
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toxicity.
Materials and Methods
Virtual screening.
The SPECS database (http://www.specs.net) is a compound library of 199,970
single-synthesized, well-characterized small molecules. This database has been widely
used in the study of compound screening processes and drug discovery and was used as
the ligand database in this study. To refine the database to include compounds with good
drug-like properties [(molecular weight ≤ 500, log P ≤ 5, number of hydrogen bond
donors ≤ 7, number of hydrogen bond acceptors ≤ 14), molecular solubility -7
(expressed as log S, where S is the solubility in mol/L), ADMET solubility -5 (base
10 logarithm of molar solubility as predicted by regression), ADMET solubility level
2 (assigns the molecule to one of seven solubility classes based on the ADMET
solubility value)], we filtered the database using Pipeline Pilot 7.5 (Scitegic, Inc., San
Diego, CA, USA) prior to screening. The filtered database was used for a subsequent
SHAFTS-3D ligand similarity search. The SHAFTS algorithm was implemented in the
ChemMapper web server, which is a hybrid 3D molecular similarity calculation
approach designed to combine the strength of pharmacophore matching and volumetric
overlay that exhibits satisfactory “scaffold hopping” capability against several
representative kinases and has been utilized for ligand-based virtual screening (29). As
PI stearoyl-arachidonyl acyl chains are long and flexible, a substituted compound with a
butyryl group attached to the oxygen atom of both PI side chains was used. The
structure of the substituted compound was drawn with ChemBio Office 2015, and MM2
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minimization was used to obtain the starting structure for the similarity search. Using
the substituted compound as the SHAFTS input molecule, the top 1000 molecules with
similarity scores > 1.0 were reserved, resulting in 822 candidates. The crystal structure
of human PI4KIIα (PDB entry: 4HNE (26)) was used as the receptor for molecular
docking. The docking accuracies of the GLIDE 5.5 (Glide, version 5.5, Schrödinger,
LLC, New York), Gold 5.0 (30-32) and Autodock 4.2 (33,34) programs were evaluated.
The substituent PI molecule was mixed with 50 decoy molecules and docked into the
PI-binding site using the three software programs. Because the GLIDE 5.5 software
provided the best PI substituent ranking, this program was used for subsequent
structural-based virtual screening. Docking studies in Gold 5.0 were performed using
the default genetic algorithm (GA) parameters and automatic settings with 100% search
efficiency. The kinase scoring function implemented in Gold was applied in the docking
calculations, and GA docking was set to be terminated if the best three solutions were
all within 1.5 Å root mean square deviations (RMSD) of each other. In Autodock 4.2,
the 50 decoy molecules and the PI substituent were docked to PI4KIIα using the
Lamarckian GA, and a 0.375 Å grid spacing was set for the energetic map calculations.
The number of GA runs was set to 10, and the standard precision (SP) GLIDE 5.5 mode
was used. The protein structure was prepared using the Protein Preparation Wizard
Workflow with default settings. The PAL loop and activation loop were defined as the
PI-binding site in which the docking grids were created (26). The 822 compounds were
docked into the defined binding site and ranked based on their GLIDE score (G-score).
Considering both the docking scores and binding modes, 522 compounds were selected
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for further evaluation, which were then structurally clustered into 60 clusters based on
their 2D molecular fingerprints using the Cluster Molecules module in Pipeline Pilot 7.5.
Two or three candidate molecules with relatively simple chemical structures and higher
G-scores within each structural cluster were retained. Ultimately, a total of 142
compounds were selected and purchased for the first-round biochemical assay. Based on
the structures of the 7 active compounds identified by the first-round biochemical assay,
the SHAFTS method was again utilized for scaffold hopping (29). A total of 53
candidates were purchased for the second-round biochemical assay.
Expression and purification for PI4KIIα, PI4KIIβ and PI4KIIIβ.
The constructs PI4KIIα,PI4KIIβ and PI4KIIIβ were expressed as GST fusion proteins
in Escherichia coli BL21-CodonPlus (DE3) competent cells. The cells were grown at
37°C until an OD600 of 1.0 was reached, at which point they were induced by 0.3 mM
isopropyl b-D-thiogalactoside at 16°C for 18 hours. The cells were then harvested and
resuspended in buffer containing 50 mM HEPES (pH 7.5), 1 M NaCl, 2 mM DTT, 20
mM MgCl2, 1 mM phenylmethylsulfonyl fluoride and 1 mg/ml lysozyme. Cell
suspensions were homogenized with a high-pressure cell disruptor (JN BIO) at 30,000
psi, and cell debris was removed by centrifugation at 120,000 g for 40 minutes. Proteins
were purified using a GST-affinity column (GE Healthcare), and the GST tag was
cleaved overnight at 4°C with PreScission protease (GE Healthcare) in buffer
containing 50 mM HEPES (pH 7.5), 300 mM NaCl, 0.1% Triton X-100 and 2 mM
MgCl2.
Kinases activity assay.
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The proteins used in this study were purchased from the following vendors: PI4KIIIα
from Creative BioMart (catalog no. PI4KA-161H), PI3Kα from Invitrogen (catalog no.
PV4788), PIK3Cδ from Millipore (catalog no. 14-604-K), PIK3Cβ from Millipore
(catalog no. 14-603-K), PIK3Cγ from Invitrogen (catalog no. PR8641C), AKT1 from
BPS (catalog no. 40003), AKT2 from Carna (catalog no. 01-102), and AKT3 from
Carna (catalog no. 01-103, Lot. No. 05CBS-3274). Additionally, PI4KIIα, PI4KIIβ and
PI4KIIIβ were expressed and purified as described above. The ADP-Glo kinase assay
(Promega) was used to evaluate the kinase activities of PI4KIIα, PI4KIIβ, PI4KIIIα,
PI4KIIIβ, PI3Kα, PI3Kδ, PI3Kβ and PI3Kγ according to previous reports with some
modifications(27). Briefly, 5 μl of kinase buffer containing different concentrations of
compounds (25 nM PI4KIIα, 268 nM PI4KIIβ, 83 nM PI4KIIIα, 109 nM PI4KIIIβ, 1.65
nM PI3Kα, 4.8 nM PI3Kβ, 7.6 nM PI3Kγ or 17 nM PI3Kδ) was added to each well of
the assay plate. For the PI4K assay, the buffer contained 50 mM HEPES (pH 7.5), 150
mM NaCl, 0.1% anzergent 3–14, 1 mM EDTA and 20 mM MgCl2, while the buffer for
the PI3K assay contained 50 mM HEPES (pH 7.5), 3 mM MgCl2, 1 mM EGTA, 100
mM NaCl, 0.03% CHAPS and 2 mM DTT. The kinase reaction was initiated by adding
5 μl of the substrate solution (50 μM PI(4,5)P2 and 25 μM ATP for the PI3Ks, 500 μM
PI and 25 μM ATP for the PI4Ks). The assay plates were then covered and incubated at
room temperature for 1 hour, and the reactions were stopped by adding 10 μl of
ADP-Glo reagent (Promega). After equilibration for 40 minutes, 20 μl of kinase
detection reagent (Promega) was added, and the mixture (40 μl total) was incubated for
an additional 60 minutes before reading the luminescence on a Varioskan Flash plate
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reader (Thermo Scientific).
The kinase activities of AKT1, AKT2 and AKT3 were measured by a mobility shift
assay. Briefly, 15 μl of kinase buffer (50 mM HEPES (pH 7.5), 0.0015% Brij-35)
containing different concentrations of the compounds (2.5 nM AKT1, 0.5 nM AKT2 or
0.7 nM AKT3) was added to each well of the assay plate. Then, a mixture consisting of
10 μl of FAM-labeled peptide (GL Biochem, 3 µM) and ATP (60 μM, 188 μM or 67 μM)
was added to the assay plate, which was then incubated at room temperature for 30
minutes. Finally, the reaction was stopped by adding 25 μl of stop buffer (100 mM
HEPES (pH 7.5), 0.015% Brij-35, 0.2% coating reagent, 50 mM EDTA), and the data
were collected with the Caliper instrument (Perkin Elmer).
Thermal shift assay and cellular thermal shift assay.
The thermal shift assay was performed on a 7500 Fast Real-Time PCR system (ABI).
Each reaction solution contained 5 M PI4KIIα, 5×SYPRO orange (Sigma-Aldrich) and
the test compounds in 20 µl of buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM
MgCl2), which was heated from 25 to 95°C at a 1% ramp rate. The melting temperature
(Tm) was calculated by the Boltzmann fitting method using Protein Thermal Shift
Software Version 1.1 (ABI). Each reaction was repeated three times.
The cellular thermal shift assay (CETSA) was carried out based on a previously
described protocol with minor modifications (35,36). For the in vivo assay, MCF-7 cells
were harvested and washed with PBS after the indicated treatments. All buffers were
supplemented with complete protease inhibitor cocktail, and the cell suspensions were
freeze-thawed three times using liquid nitrogen. The soluble fraction (lysate) was
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separated from the cell debris by centrifugation at 20,000 x g for 30 minutes at 4°C. For
the in vitro assay, the cell lysates were divided into two aliquots; one aliquot was treated
with PI-273, and the other was treated with the diluent DMSO (control) for 30 minutes.
The lysates were then divided into smaller (50 μL) aliquots and heated individually at
different temperatures for 3 minutes (S1000TM thermal cycler, BIO-RAD), followed by
cooling for 3 minutes at room temperature. The appropriate temperatures were
determined in preliminary CETSA experiments. The heated lysates were centrifuged at
20,000 x g for 30 minutes at 4°C to separate the soluble fractions from the precipitates.
The supernatants were transferred to new microtubes and analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blot.
Detection and quantification of cellular PI(4)P, LPA, PI3P, PI(4,5)P2, PI(3,4,5)P3.
All the cellular lipid contents were measured using the appropriate Mass ELISA kits
(Echelon Biosciences Inc., USA) according to the manufacturer's instructions. The kits
used included the PI(4)P Mass ELISA Kit (catalog no. K4000E), the LPA Mass ELISA
Kit (catalog no. K-2800S), the PI3P Mass ELISA Kit (catalog no. K-3300), the PI(4,5)P2
Mass ELISA Kit (catalog no. K-4500) and the PI(3,4,5)P3 Mass ELISA Kit (catalog no.
K-2500S). Briefly, cellular lipid extracts were analyzed for PI(4)P, LPA, PI3P, PI(4,5)P2
or PI(3,4,5)P3 using the appropriate Mass ELISA Kit described above and normalized
by the protein concentration in the cell lysate. For measurement of PI4P in tumor tissues,
lipids extracted from equal weights (approximately 10 mg) of xenograft tumors were
analyzed for PI4P using the PI4P Mass ELISA Kit described above and normalized by
the tissue weights. Statistical analysis was performed with 6 paired cases (37).
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Surface plasmon resonance (SPR)-based binding assay.
The SPR binding assays were performed on a Biacore T200 instrument (GE Healthcare)
at 25°C. The PI4KIIα protein was covalently immobilized on a CM5 chip using a
standard amine-coupling procedure in 10 mM sodium acetate (pH 5.0). The chip was
first equilibrated with HBS-EP buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM
EDTA, 0.05% (v/v) surfactant P20, 0.1%(v/v) DMSO) overnight. The compounds were
serially diluted in HBS-EP buffer and injected for 120 s (contact phase), followed by
120 s for the dissociation phase. The KD values of the tested compounds were
determined using the Biacore T200 evaluation software (GE Healthcare).
Mouse xenograft experiments.
Eight-week-old male BALB/c nude mice (Weitonglihua, Beijing, China) were allowed
to acclimate for 1 week under specific pathogen-free conditions in the animal facility of
the Institute of Biophysics at the Chinese Academy of Sciences. The mice were
subcutaneously injected with an MCF-7 cell suspension in a mixture of Matrigel and
DMEM, and treatment was initiated after 2 or 4 days. PI-273 was reconstituted in
DMSO and administered by intraperitoneal injection. Tumor volume was monitored via
digital calipering three times each week and calculated using the formula (smallest
diameter2×largest diameter)/2. All procedures involving animals and their care were
approved by the animal ethics committee of the Institute of Biophysics at the Chinese
Academy of Sciences.
Pharmacokinetics.
PI-273 was formulated in a 0.9% saline solution containing 10% DMSO and 0.9%
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Tween-80. Three male Sprague-Dawley (SD) rats were intravenously treated with 3.33
ml/kg formulated PI-273, and another three were intragastrically treated with 10 ml/kg
formulated PI-273. Blood samples were collected at 0.08, 0.16, 0.33, 0.67, 1, 1.5, 2, 3, 5
hours after the intravenous and intragastrical administrations. One 0.2-ml blood sample
was collected from each rat by retro-orbital bleeding. Blood was collected into
heparin-containing tubes, and plasma was obtained by centrifugation at 4,000 rpm for
10 minutes. All protocols were based on standard operating procedures approved by the
Institutional Animal Care and Use Committee (IACUC) of the Shanghai Institute of
Materia Medica at the Chinese Academy of Sciences. The plasma concentrations of
PI-273 were quantitated by the LC-MS/MS method, and noncompartmental analysis
using Phoenix 1.3 (Pharsight, USA) was performed for all analytical measurements. The
trapezoidal method was used to calculate the area under the concentration-time curve
(AUC), where AUC0-∞ = AUC0-t + Ct/ke (ke is the elimination rate constant). The
mean residence time [(MRT) = AUMC/AUC] and the elimination half-life [(t1/2) =
0.693/ke] were also calculated.
Statistical analysis.
Statistical analyses were performed using two-tailed paired Student’s t-test.
Differences were considered statistically significant when the P-value was <0.01, as
indicated in the legends. All data are expressed as the means ± S.D.
Results
Identification of PI-273 as a PI4KIIα inhibitor based on virtual screening. In this
study, virtual screening was employed to identify promising hits (Fig. 1A). The SPECS
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database (https://www.specs.net), containing 199,970 compounds, was used as the
ligand database, and compounds with unfavorable physicochemical properties were
filtered using Pipeline Pilot 7.5. The remaining 89,467 compounds were used in a
SHAFTS-3D ligand similarity search with a PI-substituted compound as the input
molecule, resulting in 822 candidates. The 822 compounds were docked into the
PI4KIIα protein pocket near the palmitoylation insertion using the GLIDE 5.5 program.
Based on the G-score and structural clustering, 142 structurally diverse compounds
were selected and purchased for biochemical assays. We then tested the effects of these
compounds on PI4KIIα activity at 100 μM and identified 7 that exhibited greater than
60% kinase activity inhibition (Suppl. Fig. 1A). Measuring the half-maximal inhibitory
concentration (IC50) values of these compounds for kinase activity in vitro (Suppl. Fig.
1B) and cell viability in vivo (Suppl. Fig. 1C) revealed that PI-93 (Suppl. Fig. 1D)
exhibited the strongest inhibitory effect. We then further tested the effect of PI-93 on the
intracellular lipid content. As shown in Suppl. Fig. 1E, the MCF-7 cells incubated with
20 μM PI-93 for 24 hours exhibited lower PI4P levels than DMSO-treated cells, while
the LPA, PI3P, PI(4,5)P2 and PI(3,4,5)P3 levels were not significantly different in either
sample.
Based on the SHAFTS-3D ligand similarity search, another 53 candidates were
selected and purchased for the second round of screening. Six compounds with an
inhibitory effect on PI4KIIα activity stronger than that of PI-93 were selected for further
analysis (Suppl. Fig. 2A, Fig. 1A). The chemical structures of these representative PI-93
analogues are presented in Suppl. Table 1 (in addition to the 6 selected compounds, 5
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compounds that shared a common parent nucleus with PI-93 in the first-round screening
and another 3 compounds that had an inhibition effect similar to that of PI-93 in the
second-round screening have also been included) and Fig. 1B. The superimposition of
PI with these six representative active compounds are shown in Suppl. Fig. 2B. The IC50
values of the compounds of interest for PI4KIIα kinase activity in vitro and cell viability
in vivo in MCF-7 cells are presented in Suppl. Table 1.
2-(3-(4-chlorobenzoyl)thioureido)-4-ethyl-5-methylthiophene-3-carboxamide (PI-273)
had the greatest inhibitory effect of MCF-7 cell viability, and its IC50 value in the
biochemical assay was 0.47 μM. In addition, our results (Suppl. Fig. 2C) also indicated
that PI-273 had the best inhibitory effect on the cellular PI4P content among the
analogues (PI-294, PI295, PI-308), which was consistent with the analogues effects on
cell viability and AKT activity. Other PI-273 analogues, such as PI-69/107/114, that had
no effect on PI4KIIα activity also had no effect on MCF-7 cells (Suppl. Table 1). In the
PI4P content assay, PI-273 reduced the intracellular PI4P content in MCF-7 cells in a
dose- and time-dependent manner (Fig. 1C). Furthermore, as shown in Fig. 1D, PI-273
treatment could specifically inhibit PI4P production but not that of other lipids, such as
LPA, PI3P, PI(4,5)P2 and PI(3,4,5)P3. Taken together, these results suggest that PI-273 is
a structurally novel PI4KIIα inhibitor with remarkable potency against the kinase
activity of PI4KIIα.
PI-273 directly binds and reversibly inhibits PI4KIIα. To validate these potential
PI4KIIα inhibitors, direct interactions between PI4KIIα and the candidate compounds
were measured by SPR. As shown in Fig. 2A and Suppl. Fig. 3A, the interactions were
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dose-dependent. The equilibrium dissociation constant (KD) between PI-273 and
PI4KIIα was approximately 9.49 µM based on the association rate constant (kon = 2.08
×102 M
-1·s
-1) and the dissociation rate constant (koff = 2.13 ×10
-3 s
-1), whereas the KD of
PI-277 was 9.59 µM and the KD of PI-282 was 5.91 µM (these two compounds have the
best solubility among PI-273/274/275/276/282/294/295/308, listed in Suppl. Table 1).
The direct interaction between these compounds and PI4KIIα was further confirmed by
a fluorescence-based thermal shift assay; PI-273 dose-dependently shifted the melting
temperature (Tm) of PI4KIIα by more than 4°C before reaching a plateau. This effect
was also observed in samples treated with PI-93, PI-277 and PI-294, whereas PI-69 did
not affect the PI4KIIα Tm value (Fig. 2B, Suppl. Fig. 3B), consistent with their
individual potencies for PI4KIIα inhibition. To evaluate the in vivo interaction between
PI-273 and PI4KIIα, we conducted a CETSA (35). The CETSA, developed by D.
Molina Martinez and his colleagues, is used for assessing target engagement by drugs in
vivo. The CETSA is based on the biophysical principle of the ligand-induced thermal
stabilization of target proteins, and it will likely become a valuable tool for validating
and optimizing drug target engagement (35,36). As shown in Fig. 2C, intact MCF-7
cells treated with 1 μM PI-273 for 96 hours and cell lysate treated with 20 μM PI-273
for 30 minutes exhibited shifted melting curves for PI4KIIα. Under DMSO treatment,
50% of PI4KIIα was degraded at 60°C, whereas in the PI-273-treated samples, 50%
degradation required temperatures of 70°C (intact cells) and 75°C (cell lysate). Thus,
PI-273 inhibits PI4KIIα activity via a direct binding event. We then assessed the
reversibility of the binding between PI-273 and PI4KIIα. PI4KIIα was preincubated
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17
with varying concentrations of PI-273 (0-1 M) for different lengths of time (0–60
minutes) before the kinase reaction was initiated. Only the concentration of PI-273 and
not the incubation time influenced the inhibitory effect of PI-273 on PI4KIIα activity
(Fig. 2D), suggesting that the interaction between PI-273 and PI4KIIα is reversible. As
further confirmation, we evaluated the enzyme kinetics of PI4KIIα, which revealed that
the slope of the velocity-to-enzyme concentration decreased with increasing PI-273
concentration (Fig. 2E), thus indicating that PI-273 is a reversible inhibitor of PI4KIIα.
Taken together, these results strongly indicate that PI-273 directly interacts with
PI4KIIα in a reversible manner.
PI-273 is a PI-competitive inhibitor of PI4KIIα. There are three types of reversible
inhibitors: competitive, uncompetitive and anticompetitive. As described above, PI-273
is a reversible inhibitor of PI4KIIα, and enzyme kinetic analysis was performed to
determine the exact type of this inhibition (Fig. 3A and B). At varying concentrations of
ATP, increasing PI-273 decreased the maximum velocity (Vmax) of the reaction (Fig. 3A
and C), but the apparent affinity (KM, Michaelis-Menten constant) of ATP for the
binding site did not change (Fig. 3A and D), indicating that PI-273 is an
ATP-uncompetitive inhibitor. However, when we varied the concentration of PI, the
Vmax of the reaction remained unchanged (Fig. 3B and C), whereas the KM value for PI
at the binding site increased, indicating that PI-273 is a PI-competitive inhibitor (Fig.
3B and D). We next performed experiments with PI4KIIα mutants to verify this
conclusion (Fig. 3E and Suppl. Table 2). As expected, the Tm of wild-type PI4KIIα was
significantly elevated in the presence of PI-273. However, mutations or deletions in the
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18
PI-binding regulation region (for example, the palmitoylation insertion, Δ165-173, or
K165E/K168E/K172E), but not those at the nucleotide-binding site (for example,
K152A, D346A), reduced the ΔTm value. We previously reported that the palmitoylation
insertion modulates kinase activity by tuning the PI-binding pocket (26,38), and these
results confirm that PI-273 is a PI-competitive inhibitor of PI4KIIα.
PI4KIIα is required for the inhibitory effect of PI-273. The selectivity of kinase
inhibitors is crucial to avoid side effects caused by off-target inhibition (39). To validate
the subtype-specific inhibitory effect of PI-273 on PI4KIIα, we tested the inhibitory
effects of PI-273 on 11 related kinases, including four PI4K subtypes (PI4KIIα, PI4KIIβ,
PI4KIIIα and PI4KIIIβ), four PI3K subtypes (PI3Kα, PI3Kβ, PI3Kγ PI3Kδ) and
three AKT subtypes (AKT1, AKT2 and AKT3). As shown in Fig. 4A, the IC50 values of
PI-273 for PI4KIIα were 0.47 µM, consistent with our previous results. The PI-273 IC50
values were much higher for the other kinases than for PI4KIIα, even for the isoform
having the greatest homology to PI4KIIα, PI4KIIβ, which had a PI-273 IC50 value more
than 30-fold higher than that for PI4KIIα. For the other PI4K subtypes (PI4KIIIα and
PI4KIIIβ), whose structures are more similar to those of PI3Ks, the IC50 values were
both more than 100 µM. Although the PI-273 IC50 value for PI3Kβ was only 10-fold
higher than that for PI4KIIα, our results shown in Fig. 1D indicate that PI-273 treatment
cannot reduce PI3P or PI(3,4,5)P3 content, suggesting that PI3Kβ is not a major target of
PI-273 in vivo. All the above results indicate that PI-273 is highly specific for PI4KIIα.
We next tested the specificity of PI-273 for PI4KIIα in vivo. We carried out
genome-scale CRISPR-Cas9 knockout screening for PI-273-resistant genes (Suppl. Fig.
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19
4A). Suppl. Fig. 4B shows that PI4KIIα is the most significantly enriched target, whose
function loss can confer strong protection to MCF-7 cells against PI-273. We then
generated PI4KIIα knockout MCF-7 cells using the CRISPR-Cas9 method (Suppl. Fig.
5). As expected, obtaining MCF-7 cells in which PI4KIIα was totally knocked out
proved to be difficult. Among 200 individual monoclones, only one PI4KIIα knockout
cell line was selected, and its sequencing results indicated that PI4KIIα exists as a
multicopy gene in MCF-7 cells (Suppl. Fig. 5A). We then further confirmed the
knockout effects of this clone by western blot analysis (Suppl. Fig. 5B) and
immunofluorescence staining (Suppl. Fig. 5C). In the cell viability assay, the PI4KIIα
knockout greatly decreased the sensitivity to PI-273. Compared to the DMSO-treated
wild-type MCF-7 cells, 10 µM PI-273 could reduce the cell viability by approximately
80%, while this ratio for the PI4KIIα knockout MCF-7 cells was only 25% (Fig. 4B),
indicating that PI4KIIα is essential for the effect of PI-273 on cell viability. Because our
previous works(4,17) and other studies indicated that PI4KIIα can regulate the AKT
signaling pathway(3), we tested the effects of our compounds on AKT signaling. As
shown in Fig. 4C and Suppl. Fig. 6A, PI-273 could suppress the AKT signaling pathway
in a dose- and time-dependent manner. However, other analogues, such as PI-294
(Suppl. Fig. 6B), PI-250 (Suppl. Fig. 6C), PI-295 and PI-308 (Suppl. Fig. 6D), had
much weaker effects on this signaling pathway, consistent with their effects on cell
viability and PI4P content. These results indicate that PI4KIIα is the target for PI-273.
We next addressed whether PI4KIIα is solely essential for the effects of PI-273 in vivo
using PI4KIIα knockout cells, and we found that PI-273 reduced the PI4P content by 40%
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20
in wild-type (CTR) cells but had no effect on PI4P in PI4KIIα knockout (KO) cells. By
contrast, PI-273 had no effect on other lipid (LPA, PI3P, PI(4,5)P2 or PI(3,4,5)P3)
content in wild-type cells or PI4KIIα knockout cells (Fig. 4D). Consistent with this
result, PI-273 decreased the p-AKT levels by 50% in wild-type cells but had no effect
on their phosphorylated levels in PI4KIIα knockout cells (Fig. 4E). In addition, PI-273
did not affect the PI4P level (Suppl. Fig. 7A) or the AKT signaling pathway (Suppl. Fig.
7B) in the PI4KIIα knockdown cells generated by siRNA. Taken together, the in vitro
and in vivo experimental results indicate that PI-273 is a PI4KIIα-specific inhibitor.
Effect of PI-273 on breast cancer cells. Compounds PI-273, PI-274, PI-277, PI-294,
PI-295 and PI-308 inhibited PI4KIIα activity both in vitro and in vivo and had varying
effects on the viability of MCF-7 cells. PI-273 had the greatest effect on PI4KIIα
inhibition and was thus selected for further exploration. To examine the biological
effects of PI4KIIα inhibition in greater detail, we tested the inhibitory effect of PI-273
on different breast cancer cell lines: MCF-7, T-47D, SK-BR-3, BT-474, MDA-MB-468,
MDA-MB-231 (KRas mutant), SUM229PE (KRas mutant), SUM159PT (HRas mutant)
and Hs 578T (HRas mutant)(40). The characteristics of the cell lines used in this study,
including the statuses of the primary tumor, origin, estrogen receptor (ER), progesterone
receptor (PR), human epidermal growth factor receptor 2 (HER-2), Ras, mice
tumorigenicity and the PI-273 IC50 value, are summarized in Suppl. Table 3. As shown
in Fig. 5A, Ras-mutant breast cancer cell lines are less sensitive to PI-273, as the PI-273
IC50 values for MCF-7, T-47D, SK-BR-3, MDA-MB-468 and BT-474 cells were 3.5 µM,
3.1 µM, 2.3 µM, 3.9 µM and 2.1 µM, respectively, while the IC50 values for all the
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Ras-mutant cell lines were higher than 10 µM. The 56-carboxyfluorescein diacetate
succinimidyl ester (CFDA SE) assay was used to evaluate the effects of PI-273 on cell
proliferation. As shown in Suppl. Fig. 8A, 1 µM and 2 µM PI-273 inhibited the cell
proliferation of both MCF-7 and T-47D cells in a time-dependent manner, but these
PI-273 doses did not influence the proliferation of Hs 578T cells. These results are
consistent with the inhibition of these compounds against PI4KIIα in different cell lines
and indicate that MCF-7 and T-47D cells are much more sensitive to PI-273. Cell cycle
regulation by PI-273 was examined by 3,8-diamino-5-[3-(diethylmethylammonio)
propyl]-6-phenylphenanthridinium diiodide staining and flow cytometry. Interestingly,
PI-273 blocked the cell cycle at the G2/M phase (Fig. 5B), similar to the effect of
PIK-75, another substrate-competitive inhibitor of PIKs (PI3Kα)(41). The effect of
PI-273 on cell apoptosis was evaluated by a terminal deoxynucleotidyl transferase dip
nick end labeling (TUNEL) assay. As shown in Fig. 5C, treatment with PI-273 induced
cell apoptosis in all three Ras wild-type breast cancer cells: MCF-7, T-47D and
SK-BR-3. In addition to the cell viability assay, the inhibitory effects of PI-273 on the
tumorigenicity of cells in vitro were evaluated with plate clone-forming tests (Fig. 5D)
and soft agar clone-forming tests (Fig. 5E). PI-273 exhibited prominent drug potency in
MCF-7 and T-47D cells (1 µM PI-273 reduced both plate colony and soft agar colony
growth by more than 50%). However, Hs 578T and MDA-MB-231 cells were less
sensitive to PI-273 than MCF-7 and T-47D cells. To assess whether sensitivity is
dependent on PI4KIIα expression in different cell lines, the PI4KIIα expression levels
(Suppl. Fig. 8B) and PI4P regulation effects (Suppl. Fig. 8C) were measured. The
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results showed that all the cell lines had high PI4KIIα expression levels, and the PI4P
content could be regulated by PI-273 in all four cell lines tested. Although the PI4KIIα
expression levels were different among these cell lines, such differences could not
distinguish the sensitivity of PI-273. However, the cell sensitivities to PI-273 showed
strong correlation with the Ras status (Fig. 5 A-E and Suppl. Table 3.). Taken together,
these results indicate that PI-273 retards cell proliferation by blocking cells at the G2/M
phase, inducing cell apoptosis and suppressing cell tumorigenicity, and these effects are
dependent on the cell type.
Antitumor effect of PI-273 in a xenograft model. To determine whether PI-273 can
suppress breast cancer in vivo, we engrafted MCF-7 cells, which were confirmed to be
sensitive to PI-273 in the above experiments, into the right flank region of BALB/c
nude mice. Four days after cell injection, the mice were randomized and received either
an intraperitoneal injection of PI-273 25 mg/kg/day or vehicle (5% DMSO). The mice
were sacrificed 24 hours after the 15th
injection. PI-273 profoundly suppressed the
tumor volume (Fig. 6A) and weight (Fig. 6B) in the MCF-7 xenografts compared to the
effects of treatment with the vehicle. Another group of parallel experiments indicated
that an intraperitoneal injection of PI-273 at 50 mg/kg/2 days also inhibited MCF-7
xenograft tumor growth, although with lower efficiency than the 25 mg/kg/day injection
(Suppl. Fig. 9A and B). We also assessed the toxic effects of PI-273 in mice, and no
lethargy, weight loss (Fig. 6C) or other physical indicators of sickness were observed.
Various tissues, including liver, intestine, lung, kidney, spleen and stomach tissue, were
examined by hematoxylin eosin (HE) staining, and no tissue damage (macroscopic or
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microscopic) was observed (Suppl. Fig. 9C). These studies establish the effectiveness
and safety of PI-273 for antitumor applications. We further assessed the utility of PI-273
in animal experiments. SD rats were treated with 0.5 mg/kg PI-273 intravenously or 1.5
mg/kg PI-273 intragastrically. Blood samples collected retro-orbitally were collected at
0.08, 0.16, 0.33, 0.67, 1, 1.5, 2, 3, 5 hours after the intravenous and intragastrical
administrations. The plasma levels were analyzed by LC-MS/MS, and the summarized
pharmacokinetic results are presented in Table 1. Compound PI-273 is retained in rats at
a half-life of 0.411 hours for intravenous administration and 1.321 hours for
intragastrical administration, and the absolute bioavailability of PI-273 is 5.1%. These
pharmacokinetic results indicate that PI-273 as lead compound showed moderate
pharmacokinetic activities. Histological examination using the TUNEL assay to detect
apoptotic cells revealed a significant increase in the number of apoptotic cells in the
PI-273-treated MCF-7 xenografts compared to those treated with vehicle (Fig. 6D). A
significant decrease in proliferation, measured by Ki-67 staining, was observed in the
PI-273-treated MCF-7 xenografts compared to the vehicle (Fig. 6E). The PI4P content
and p-AKT levels in MCF-7-induced tumors were measured to evaluate the effect of
PI-273 in the xenograft model. Consistent with the data shown above, the PI-273-treated
tumors exhibited reduced PI4P content and p-AKT levels (Fig. 6F). These results
confirm that the PI4KIIα-specific SMI PI-273 suppresses proliferation, survival, and
AKT signaling in MCF-7-induced breast cancer in vivo. The mechanism underlying the
PI-273 antitumor effect is summarized in Fig. 6G; PI-273 inhibits the kinase activity of
PI4KIIα, leading to reduction of the intracellular PI4P content and suppression of the
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PI3K/AKT signaling pathways, resulting in retarded cell proliferation and increased
apoptosis, and, consequently, tumor growth inhibition. PI-273 shows good potential for
use in clinical trials for human breast cancer.
Discussion
Because PI4KIIα plays important roles in Golgi trafficking(14,42,43) and tumor
progression(3,4,44), SMIs of PI4KIIα have potential as chemical tools for studying the
biological function of PI4KIIα and breast cancer treatment(10,19,42). Here, PI-273 was
screened out and identified as the most potent inhibitor of PI4KIIα activity and breast
cancer cell viability. Biochemistry and kinetic analyses revealed that PI-273 directly
binds and reversibly inhibits PI4KIIα. The fact that PI-273 is a substrate-competitive
inhibitor rather than an ATP-competitive inhibitor is its greatest advantage, as this
results in kinase isoform selectivity. Further study confirmed that PI4KIIα is essential
for the effects of PI-273. As the first substrate-competitive inhibitor in the PI4K family
and the third such inhibitor in the entire PIK family (PIK-75 is a substrate-competitive
inhibitor of PI3K(23) and NIH-12848 is a substrate-competitive inhibitor of
PI5P4Kγ(25)), PI-273 is highly isoform-selective and has significant anti-breast cancer
effects without toxicity, showing its great potential for basic PI4KIIα and
pharmaceutical research.
Numerous small molecules have been previously identified as PIK inhibitors, as
summarized in Supplemental Table 4, and none affect PI4KIIα, except for EGCG(45),
resveratrol(46) and adenosine(47). In fact, we tested the effects of all the potential
candidates on PI4KIIα activity prior to screening, including EGCG, resveratrol,
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adenosine, PI3K inhibitors (Wortmannin, LY294002, ZSTK474), a PI3Kα inhibitor
(PI103), a PI3Kδ inhibitor (IC87114), a PI3Kγ inhibitor (AS605240), and a PI4KIIIβ
inhibitor (PIK93) (Suppl. Fig. 10). As observed previously, PI4KIIα was insensitive to
PI3K and type III PI4K inhibitors(10,48,49), and the IC50 values of both resveratrol and
EGCG were greater than 100 μM. Moreover, these molecules are all broad-spectrum
inhibitors and thus cannot be used for PI4KIIα subtype-specific functional studies.
Therefore, screening subtype-specific inhibitors for PI4KIIα is of great significance.
As summarized in Supplemental Table 4, hundreds of small molecules were
identified as PIK inhibitors by screening, but most were ATP-competitive, except for
PIK-75(23) and NIH-12848(25), limiting their potential for high isoform selectivity.
The lack of structural information regarding the PIK substrate complex is a barrier to
developing substrate-competitive inhibitors because lipid substrate binding requires the
kinase to adopt a membrane-bound conformation that is likely different from the
structure observed in crystal form. We have recently overcome this limitation by
combining data generated from crystal structure, molecular docking and biochemical
studies to identify the putative PI-binding pocket of PI4KIIα. We determined that a
PI4KIIα-unique insertion, the palmitoylation insertion, affects the conformation of the
substrate-binding pocket and thus regulates its kinase activity(26). In this study, we
found that PI-273 can directly bind the palmitoylation insertion and functions as a
PI-competitive reversible inhibitor (Fig. 2D, 2E, 3A, 3B and Table 1). These results also
confirm our previous MD simulation results that the palmitoylation insertion influences
substrate binding. Our work suggests that MD simulation may be helpful for identifying
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26
PIK substrate-binding pockets, thus facilitating substrate-competitive inhibitor
screening. However, with respect to the PI-competitive mechanism of PI-273,
determining whether PI-273 can directly bind the substrate pocket or only induce
allosteric regulation of the substrate-binding site is difficult because of the lack of
inhibitor-soaked crystal structure information. Additional efforts to elucidate the binding
mechanism of PI-273 will be made in the future using NMR, crystal structure, hydrogen
deuterium exchange (HDX) and single-molecule fluorescence resonance energy transfer
(FRET) analysis.
In this study, we demonstrated that PI-273 can inhibit breast cancer cell
proliferation (Suppl. Fig. 8A), block the cell cycle (Fig. 5B) and induce cell apoptosis
(Fig. 5C). We then further confirmed its effects on colony formation (Fig. 5D and 5E)
and on an MCF-7 cell-induced xenograft model (Fig. 6A and 6B), all of which indicated
the suppressive effect of PI-273 on breast cancer growth both in vitro and in vivo.
Because previous studies from our group and others have suggested that in addition to
breast cancer, many other cancers, such as malignant melanoma and thyroid carcinoma,
also feature high PI4KIIα expression(4,16), we suspect that PI-273 may have inhibitory
effects on these types of tumors. In our experiment, we also tested the effects of PI-273
on MDA-MB-435 cells, which have been identified as malignant melanoma
cells(50-52). To our surprise, the MDA-MB-435 cells were even more sensitive to
PI-273 in both the plate clone-forming test and the soft agar clone-forming test
compared to the MCF-7 and T-47D cells (data not shown), suggesting that PI-273 may
suppress melanoma growth and merits further investigation.
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Because precision medicine is considered the optimal means of completely
conquering cancer, clarifying which patient categories are sensitive to particular drugs is
important. In our study, Hs 578T, MDA-MB-231, SUM229PE and SUM159PT cells
were relatively resistant to PI-273 in terms of cell viability, cell cycle and apoptosis (Fig.
5), which is consistent with previous studies concluding that monotherapies targeting
the PIK signaling pathway are largely disappointing for Ras-mutant cancers(53).
PI3K/AKT and Ras/MEK/ERK are two crucial interlinked growth and survival
signaling pathways in tumors[52], and Torbett et al. indicated that Hs 578T cells with
activated forms of the H-Ras oncogene resulting in the activation of parallel pathways
that are able to compensate for the loss of PI3K signals. Different groups have indicated
that PI3K inhibitor treatment alone does not result in the regression of Ras -mutant lung
tumors(54,55), pancreatic cancer(56) or non-small cell lung cancer(57) because of
Ras/MEK/ERK-dependent signaling activation. However, compared to monotherapies,
combining the two treatments (MEK and PI3K inhibitors) resulted in a significant
survival advantage in these cancers. Taken together, these results suggest that the
pharmacodynamic effects of PI-273 may also be related to Ras activity, which is another
interesting topic to be explored in future work.
Interestingly, because PI-273 treatment and PI4KIIα knockout had negligible
effects on the PI(4,5)P2 and PI(3,4,5)P3 levels (Fig. 1D and Fig. 4D), we hypothesize
that the effect of PI4KIIα inhibitors on AKT activity does not result from the direct
regulation of the PI(4,5)P2 and PI(3,4,5)P3 levels. Instead, numerous studies have
indicated that PI4KIIα is important for receptor activity via trafficking
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28
regulation(13,17,58,59). Therefore, it is possible that the regulation of PI4KIIα
inhibitors on AKT activity is dependent on its receptor regulation effect, but this
requires further validation.
In summary, we identified PI-273 as a lead substrate-competitive inhibitor of
PI4KIIα based on a unique insertion, the palmitoylation insertion, of PI4KIIα as a
binding site. Our results also demonstrate the significance, safety, and efficacy of
PI4KIIα as a therapeutic target, and PI-273 will be optimized as a therapeutic agent for
cancer in the future.
Note: Three Supplementary data files accompany this manuscript on the Cancer
Research website. Suppl. data 1 including Suppl. method .Suppl. data 2 incuding 4
tables: Suppl. Table 1. Chemical structures and activity of representative PI-93
analogues; Suppl. Table 2. Activity and △Tm of the PI4KIIα mutants;Suppl. Table 3.
Cell lines used in this study; Suppl. Table 4. Reported PIK inhibitors. Suppl. data 3
incuding 10 figures:Suppl. Fig. 1 First-round screening of PI4KIIα inhibitors;Suppl. Fig.
2 Second-round screening of PI4KIIα inhibitors;Suppl. Fig.3. Direct interaction
between PI-273 analogs and PI4KIIα;Suppl. Fig. 4. PI-273 resistance screens using
CRISPR-Cas9;Suppl. Fig.5. Generate and identify PI4KIIα knock out MCF-7
cells;Suppl. Fig. 6. Effects of PI-273 and its analogs on PI4KIIα downstream signaling
pathways;Suppl. Fig. 7. PI4KIIα is required for the regulation of PI-273 on signaling
transduction in breast cancer cells;Suppl. Fig. 8. PI-273 inhibits the proliferation of
breast cancer cells.;Suppl. Fig. 9 Anti-tumor effect of PI-273 in an MCF-7-induced
xenograft model;Suppl. Fig. 10 Inhibitory effect of 100 μM PIK inhibitors on PI4KIIα.
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29
Acknowledgments: We thank Pietro De Camilli for providing the PI4KIIα antibody
and Shane Minogue for providing the full-length human PI4KIIα cDNA.
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33
Table 1. Pharmacokinetical results of the SD rats after intravenous administration
of 0.5 mg/kg PI-273 and intragastrical administration of 1.5 mg/kg PI-273
respectively.
Pharmacokinetical parameters
PI-273
Intravenously(0.5 mg/kg) Intragastrically(1.5 mg/kg)
Mean ± SD Mean ± SD
Cmax (ng∙ml-1
) 2392.7 ± 46 116 ± 23
Tmax (h) 0.083 ± 0.00 0.278 ± 0.096
t1/2 (h) 0.411 ± 0.087 1.321 ± 0.657
MRT0-12 (h) 0.23 ± 0.072 1.25 ± 0.061
MRT0-∞ (h) 0.23 ± 0.072 1.571 ± 0.378
CL/F (L∙h-1
∙kg-1
) 0.649 ± 0.204 11.4 ± 1.32
Vd/F (L∙kg-1
) 0.38 ± 0.118 21.0 ± 7.86
AUC0-12 (ng∙h∙ml-1
) 816 ± 218 125 ± 8.28
AUC0-∞ (ng∙h∙ml-1
) 816 ± 218 132 ± 16.0
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34
Figure legends
Figure 1 Identification of PI4KIIα small-molecule inhibitors. (A) Schematic
representation of the virtual screening strategy adopted in this study. (B) Chemical
structures of PI-273 and PI-69. (C) The effect of PI-273 on cellular PI4P content.
MCF-7 cells were treated with DMSO or the indicated concentrations of PI-273 for the
indicated times. The cells were then harvested for PI4P measurement. (D) MCF-7 cells
were treated with DMSO or 1 μM PI-273 for 24 hours before being collected for the
indicated lipids analyses. The values are presented as the means ± S.D. from three
independent experiments, and all the above experiments were performed three times in
triplicate. * indicates p<0.01.
Figure 2 PI-273 directly binds and reversibly inhibits PI4KIIα. (A) Representative
SPR binding curves for PI4KIIα and 2-fold serial dilutions of PI-273 from 1.25 μM to
20 μM. (B) Thermal shift assay indicating the stabilization of PI4KIIα by PI-273.
Melting curves for 5 μM PI4KIIα protein in the presence of varying concentrations of
PI-273 or PI-69. (C) A cellular thermal shift assay was performed to evaluate the
interaction between PI-273 and PI4KIIα in intact cells (in vivo) and cell lysates (in
vitro). (D) PI4KIIα was preincubated with the indicated concentrations of PI-273 (0-1
μM) for different durations (0–60 minutes) before ATP was added. The graphs represent
normalized % inhibition compared to preincubation time. (E) The initial velocity
kinetics compared to varying PI4KIIα enzyme concentrations (2.5-10 ng/μl) at the
indicated concentrations of PI-273 (0-2 μM). All the above experiments were performed
three times with comparable results.
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35
Figure 3 PI-273 is a substrate-competitive inhibitor of PI4KIIα. Enzyme kinetics
analysis of PI4KIIα inhibition by PI-273. Activity assays were performed with varying
concentrations of (A) ATP and (B) PI. Double-reciprocal plots of initial velocities
(Lineweaver-Burk plots) showing uncompetitive inhibition by PI-273 toward ATP and
purely competitive inhibition toward PI as a substrate. The experiment was performed
three times with comparable results. (C) Vmax and (D) KM values for ATP and PI under
varying concentrations of PI-273. (E) Thermal shift assay demonstrating the
stabilization of PI4KIIα variants by PI-273. PI4KIIα (5 μM) variants were treated with
PI-273 (50 μM) and then subjected to the thermal shift analysis. The values are
presented as the means ± S.D. from three independent experiments (Student's t-test),
and this experiment was performed three times in triplicate. * indicates p<0.01 and #ns
indicates no difference.
Figure 4 PI-273 inhibits PI4KIIα downstream signaling transduction in breast
cancer cells. (A) PI-273 IC50 values for PI4KIIα, PI4KIIβ, PI4KIIIα, PI4KIIIβ, PI3Kα,
PI3Kδ, PI3Kβ, PI3Kγ, AKT1, AKT2 and AKT3. (B) The anti-proliferation activity of
PI-273 in wild-type MCF-7 cells and PI4KIIα knockout MCF-7 cells. (C) Specific
regulation of AKT signaling by PI-273 in MCF-7 cells. MCF-7 cells were treated with
the indicated doses of PI-273 for 3 days, followed by treatment with 100 ng/ml EGF for
10 minutes. AKT phosphorylation and protein expression levels were measured by
western blot. The effects of PI-273 on (D) LPA, PI4P, PI3P, PI(4,5)P2, PI(3,4,5)P3
content and (E) AKT signaling in wild-type MCF-7 cells (CTR cells) and PI4KIIα
knockout MCF-7 cells (KO cells). Cells were treated with 1 μM PI-273 or DMSO for
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24 hours (for lipid ratio detection) or 3 days (for AKT signaling detection), respectively.
Lipid contents were detected by the appropriate Mass ELISA Kit. AKT phosphorylation
and protein expression levels were measured by western blot. All the values are
presented as the means ± S.D. from three independent experiments, and all the above
experiments were performed three times in triplicate. * indicates p<0.01 and #ns
indicates no difference.
Figure 5 PI-273 inhibits the proliferation of breast cancer cells. (A) Cell viability
assays (WST-8 assay) were performed in MCF-7, T-47D, SK-BR-3, BT-474,
MDA-MB-468, MDA-MB-231, SUM229PE, SUM159PT and Hs 578T cells after
treatment with the indicated concentrations of PI-273 for 72 hours. (B) Six breast cancer
cells were treated for 48 hours with DMSO or 2 μM PI-273 and then labeled with PI.
Cells were analyzed by flow cytometry. The graph shows the % of cells in each cell
cycle phase. (C) Apoptosis ratio in cells treated with PI-273. Cells were counted using a
TUNEL kit after being treated with DMSO or 2 μM PI-273 for 48 hours. The % of
apoptotic cells are presented on the y-axis. (D) and (E) Effects of PI-273 on colony
formation in MCF-7, T-47D, Hs 578T and MDA-MB-231 cells. Anchorage-dependent
cell growth and anchorage-independent cell growth were measured using the plate
clone-forming assay and the soft agar clone-forming assay, respectively. Colony
numbers were determined after 7 days of incubation for the plate clone-forming assay
and 14 days for the soft agar clone-forming assay. Scale bar: 200 μm. All the values are
presented as the means ± S.D. from three independent experiments (Student's t-test),
and all the above experiments were performed three times in triplicate * indicates
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p<0.01 and #ns indicates no difference.
Figure 6 PI-273 suppresses breast cancer in vivo. (A) Tumor growth curve, (B) tumor
weight and (C) mouse weight for MCF-7 xenografts in BALB/c nude mice treated with
25 mg/kg/day PI-273 or an equal volume of vehicle for 16 consecutive days. Statistical
analysis was performed using the paired t-test. (D) TUNEL+ staining of the histological
sections of MCF-7 xenografts. The y-axis represents the ratio of TUNEL+
cells per ten
fields for one tumor sample (n = 5 tumors/treatment). Scale bar: 20 μm. (E) Ki-67
immunohistochemistry staining of xenograft MCF-7 tumor sections. The y-axis
represents the ratio of Ki-67+ cells per ten fields for one tumor sample (n = 5
tumors/treatment). Statistical analysis was performed using Student’s t-test. Scale bar:
100 μm. (F) Effect of PI-273 on the phosphorylation levels of AKT and the PI4P content
in MCF-7 xenograft tumors. All the values are presented as the means ±S.D. from the
indicated samples. * indicates p<0.01. (G) Model of the antitumor effect of PI-273.
PI-273 can specifically bind PI4KIIα, inhibiting its activity and, notably, reducing the
cellular PI4P content, thus resulting in suppressed PI3K/AKT signaling transduction.
This activity blocks cells at the G2/M phase, resulting in proliferation arrest and
apoptosis. Together, these effects can retard the growth of breast tumors.
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Published OnlineFirst August 21, 2017.Cancer Res Jiangmei Li, Zhen Gao, Dan Zhao, et al.
, inhibits the growth of breast cancer cellsαinhibitor of PI4KIIPI-273, a substrate-competitive, specific small molecule
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