Unexpected Discovery of Dichloroacetate Derived Adenosine Triphosphate Competitors Targeting Pyruvate Dehydrogenase Kinase to Inhibit Cancer Proliferation

Article abstract of DOI:10.1021/acs.jmedchem.5b01828

Pyruvate dehydrogenase kinases (PDKs) have recently emerged as an attractive target for cancer therapy. Herein, we prepared a series of compounds derived from dichloroacetate (DCA) which inhibited cancer cells proliferation. For the first time, we have successfully developed DCA derived inhibitors that preferentially bind to the adenosine triphosphate (ATP) pocket of PDK isoform 1 (PDK1).

Full text of DOI:10.1021/acs.jmedchem.5b01828

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Brief Article
Unexpected discovery of dichloroacetate derived
adenosine triphosphate competitors targeting pyruvate
dehydrogenase kinase to inhibit cancer proliferation
Shao-Lin Zhang, Xiaohui Hu, Wen Zhang, and Kin Yip Tam
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01828 • Publication Date (Web): 23 Mar 2016
Downloaded from http://pubs.acs.org on March 25, 2016
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Journal of Medicinal Chemistry
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Unexpected discovery of dichloroacetate derived adenosine triphos-
phate competitors targeting pyruvate dehydrogenase kinase to in-
hibit cancer proliferation
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ShaoꢀLin Zhang, # Xiaohui Hu, # Wen Zhang and Kin Yip Tam *
Drug Development Core, Faculty of Health Sciences, University of Macau, Macau, China
ABSTRACT: Pyruvate dehydrogenase kinases (PDKs) have recently emerged as an attractive target for cancer therapy. Herein, we
prepared a series of compounds derived from dichloroacetate (DCA) which inhibited cancer cells proliferation. For the first time,
we have successfully developed DCA derived inhibitors that preferentially bind to the adenosine triphosphate (ATP) pocket of PDK
isoform 1 (PDK1).
INTRODUCTION
late the kinase activity by binding to the ATP pocket. DCA, a
structural analogue of PDC substrate pyruvate, binds to the
pyruvateꢀbinding site in the center of the R domain to regulate
Most cancer cells feature a switch in metabolism from mitoꢀ
chondrial oxidative phosphorylation to cytoplasmic aerobic
glycolysis even under normoxia conditions. Such metabolic
remodeling is characterized by increased glycolysis and supꢀ
pressed glucose oxidation, which provides cancer cells with
proliferative advantages. 2ꢀ4 Disruption of this unique metabolꢀ
ic pathway of cancer cells represents therapeutic opportunities
in anticancer treatment. 5, 6
22
23
1
the activity of PDKs. In 2007, Michelakis et al first reꢀ
ported that DCA could inhibit the growth of cancer cells.
Since then, DCA has become a hot molecule and received an
increasing attention. However, its low binding affinity to
PDKs requires a high dose to become efficacious, which apꢀ
peared to limit its clinic application. Moreover, the clinical
records suggested that patients receiving longꢀterm DCA
treatment showed reversible limb motor weakness and demyeꢀ
PDKs function as a molecular switch that downꢀregulates miꢀ
tochondrial respiration and enhance aerobic glycolysis via
phosphorylating pyruvate dehydrogenase complex (PDC), a
gateꢀkeeper mitochondria enzyme, converting pyruvate to
acetyl coenzyme A (acetylꢀCoA).
increase the oxidative phosphorylation by activating PDC is an
attractive therapeutic strategy to reverse the Warburg effect
and inhibit cancer cell proliferation. Recently, four PDK
24
lination of cerebral and cerebellar white matter. It is highly
desirable to modify DCA to offer more potent anticancer
agents. Recently, series of DCA derivatives have been develꢀ
oped, however, none of their action mechanisms had been
explained explicitly. 9, 25ꢀ27
7, 8
Inhibition of PDKs to
O
O
O
9
S
O
N
CF3
isoforms (PDK1, PDK2, PDK3 and PDK4) in mitochondria
have been isolated and characterized in terms of their differꢀ
ences in activity, tissue distribution and regulations. 10 Among
these isoforms, PDK1 is mostly associated with cancer maligꢀ
nancy. It had been reported that PDK1 was remarkably overꢀ
F3C
HO
N
N
N
CN
H
HO
O
Cl
O
Nov3r
AZD7545
H
Cl
N
O
NH2
Cl
O
11
ONa
O
expressed in multiple human tumor such as lung cancer,
Cl
12
13
14
DCA
head squamous cancer,
myeloma
and gastric cancer.
Pfz3
N
Cl
Cl
PDK1 is transcriptionally regulated by oncogenes cꢀMYC and
N
Cl
hypoxiaꢀinducible factor 1α (HIFꢀ1α) to control metabolic and
OH
Cl
O
15
O
malignant phenotypes of cancer cell.
Collectively, it has
O
NH
been suggested that PDK1 is a viable anticancer target. 16
O
O
HO
HO
So far, four types of inhibitors have been reported to inhibit
N
the activity of PDKs via different mechanisms. As show in
OH
O
Radicicol
VER-246608
17
18
Figure 1, Nov3r and 1 (AZD7545) are the PDK2 inhibiꢀ
tors with the same warhead, namely the trifluoromethylproꢀ
panamide group, which binds to the lipoamideꢀbinding site
located at one end of the R domain of PDKs. In contrast, Pfz3
19 binds to PDKs at the other end of R domain of the N termiꢀ
Figure 1 Structures of known PDKs inhibitors
In this paper, we prepared series of DCA analogues with the
aim to find new inhibitors with better inhibitory potency and
stronger binding interactions than that of DCA. We found that
the DCA based inhibitors bind to the ATP pocket, but not preꢀ
sumed pyruvate pocket.
20
nus opposing the lipoamideꢀbinding site. VERꢀ246608 and
Radicicol 21 are ATPꢀcompetitive inhibitors, which down reguꢀ
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RESULTS AND DISCUSSION
breast cancer cell line Michigan Cancer Foundationꢀ7 (MCFꢀ7)
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by using MTT assay. We identified three hit compounds 15,
26 and 39, which completely inhibited the growth of the canꢀ
cer cells at 40 µM. The inhibition was also confirmed by black
field pictures of the cultured cells acquired from IncuCyte
Zoom (Essen Biosciences) (see Figure S1). Compared with
DCA, 15 and 26 exhibited a very low IC50 values, about 1000ꢀ
fold more potent against A549 and MCFꢀ7. Excitingly, 39 was
found to be 3000ꢀfold more potent than DCA against these
two cell lines (see Table 1, Figure S2). We further evaluated
the inhibition rate of the compounds to normal human breast
cell MCFꢀ10a, where the compounds showed relative low
cytotoxic effects. The inhibition rate was only 10.32% for 39
at the concentration of 20 µM (see Table 1). In order to study
the mode of cell death, 15, 26 and 39 were used to induce
MCFꢀ7 apoptosis and examined by Annexin VꢀFITC/PI FACS
assay. As shown in the Figure 2, the percentages of apoptosis
for MCFꢀ7 cells treated with 15, 26 and 39 at 20 µM for 10 h
were 9.87%, 10.60% and 11.80%, respectively, which outperꢀ
formed DCA at 10 mM. When time increased to 15 h, the
apoptosis rate was 14.2% for 39, suggesting the induction of
apoptosis in MCFꢀ7 by 39 follows a timeꢀdependent manner.
Modification of DCA leads to potent compounds that inhibit
cancer cells proliferation. According to the literature, 25, 26 the
carboxyl group is the only modifiable site in DCA without
detrimental effect on its antiꢀcancer property. With this notion
in mind, we designed a chemical series by modifying the carꢀ
boxyl group to study the antiꢀproliferation potency against
cancer cells. We synthesized and purified about 50 novel DCA
analogues (see Scheme 1), whose structures were confirmed
by 1H NMR, 13C NMR and HRMS spectra (see SI).
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O
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O
DCM, Et3N
Ice bath
Cl
Cl
R
R
NH2
Cl
Cl
Cl
5-53
O
Cl
Cl O
Cl
Cl O
Cl
N
N
Cl
F3C
NH Cl
NH Cl
NH Cl
N
N
15
26
39
Cl
Cl
Scheme 1 General synthetic route for target compounds 5−53. R groups
represent as substituted aromatic rings, such as benzene, pyrimidine and
pyridine rings, etc (detailed information was listed in SI).
We then tested their antiꢀproliferative activity against the huꢀ
man lung adenocarcinoma epithelial cell line (A549) and
Table 1 In vitro activity of the selected compounds on A549 and MCFꢀ7, and human normal cell line MCFꢀ10a.
IC50 (µM) a
Inhibition rate (%) for MCFꢀ10a
Compounds
Kd (µM) b
A549
MCFꢀ7
22.89 ± 1.44
19.34 ± 1.06
3.12 ± 0.20
10 µM
40 µM
15
26
39
26.02 ± 1.15
28.42 ± 0.98
7.46 ± 0.81
7.57
41.56
NT c
4.91
42.36
NT c
3.51
3.42 (20 µM)
10.32 (20 µM)
41.10 (10 mM)
40.8
> 1000
DCA
23.58 ± 1.26 (mM)
12.35 ± 0.74 (mM)
IC50 is the 50% inhibitory concentration. b Dissociation constants (Kd) shows the binding affinity of the inhibitor to the PDK1, which were determined by
a
ITC described in experimental procedures (Figure S1). c Not test.
26 (20 µM)
15 (20 µM)
Blank
DCA (10 mM)
39 (20 µM) for 10 h
39 (20 µM) for 15 h
39 (20 µM) for 5 h
***
**
**
**
**
**
Annexin V
Figure 2 Flow cytometer analysis using FITC Annexin V apoptosis detection Kit with PI. The cells were treated with DCA, 15, 26 and 39 for different time,
and then stained with the kit. Cells in the lower right quadrant indicate Annexin Vꢀpositive/PI negative, early apoptotic cells. The cells in the upper right
quadrant indicate Annexin Vꢀpositive/PI positive, late apoptotic or necrotic cells. * P < 0.05, versus control group.
Compound 39 binds to PDK1. With the improved anticancer
activity of 39 and others, we would like to address the quesꢀ
tion whether the compound interacts with its supposed target
PDKs. PDK1 solution was titrated with 39 in an isothermal
calorimetry experiment, a moderate Kd (40.8 µM) was derived,
indicating that 39 interacts directly with PDK1 (see Figure
unambiguous evidence refers to the binding mode for DCA
analogues other than a plausible pyruvate binding pocket that
22
based on the crystal structure of DCA and PDK protein. To
rationalize the binding process, 39 was docked into the pyꢀ
ruvate pocket of PDK1 by SybylꢀXꢀ2.1. A negative total bindꢀ
ing scores (ꢀ7.81) and physical hindrance of the pocket in the
model led us to believe that it is unlikely for 39 to bind to this
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3A). PDK1 has four known binding pockets. To date, no
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Journal of Medicinal Chemistry
pocket. To explore which binding pocket(s) that 39 is likely to we turn our attention to study 15, 26 and 39 on PDC activation.
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bind, we then performed a series of competitive titration exꢀ
periments. Specifically, we titrated PDK1 with adenosine diꢀ
phosphate (ADP) (in the absence of 39, Figure 3B). The Kd
value was 4.27 µM (Ka = 234000) with a Gibbs free energy
(ꢁG) of ꢀ7325.6 J/mol. However, in the presence of 39, the Kd
value increased to 7.87 µM (Ka = 127000, Figure 3C), and
more importantly, ꢁG value dropped to ꢀ6965.2 J/mol, indicatꢀ
ing preꢀoccupation of the ATP pocket by 39. Titration of
PDK1 with 1 in the presence or absence of 39 did not show
any discrepancy in Kd or ꢁG values (Figure 3D and 3E).
Hence it is unlikely that 39 is competing with 1 for the lipoamꢀ
ideꢀbinding site. AcetylꢀCoA binding to PDK1 was undetectaꢀ
ble under the same experimental conditions. All together, theꢀ
se observations suggest that 39 binds to an unexpected ATP
pocket in PDK1. To the best of our knowledge, this is the first
report of DCA analogues binding to an ATP pocket in PDK1.
Kinetic experiments were carried out by measuring the forꢀ
mation of reduced nicotinamide adenine dinucleotide (NADH)
during the course of pyruvate conversion to acetylꢀCoA. To
our surprise, 15, 26 and 39 at 50 µM barely activated PDC
when ATP was included in the reaction mixture, while 10 mM
DCA could slightly activate PDC (see Figure 4A). Interestingꢀ
ly, in the absence of ATP, the activity of PDC was enhanced
by the addition 15, 26 and 39, suggesting these compounds
could serve as PDC activators (see Figure 4B). However, it
was still not clear whether they activate PDC directly or via
the inhibition of PDKs. To address this question, we examined
PDC activity by adding recombinant PDK1, which was able to
inhibit PDC activity completely (see Figure 4C). When 15, 26
and 39 were added to the mixture, the activity of PDC was
restored to a large extent. This observation provides a strong
evidence to suggest that 15, 26 and 39 act through PDK1 to
activate PDC. Inability of 15, 26 and 39 to activate PDC in the
presence of ATP could be explained by their relative low bindꢀ
ing affinity to PDK1 as compared with ATP. Moreover, we
found that the phosphorylation of PDC by PDK1 was inhibited
by compounds 15, 26 and 39, as well as DCA (see Fig S4),
another proof of their regulation PDC via kinase inhibition.
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Compounds 15, 26 and 39 activate PDC. Inhibition of PDK1
leads to PDC activation. Firstly, PDK1 kinase activity was
evaluated in the presence of DCA, 15, 26 and 39 using a
commercial kit (see SI). As shown in Figure S3, 15, 26 and 39
at 50 µM reduced the consumption of ATP, which was used to
phosphorylate peptide fragment around Ser293 site of PDC by
PDK1, suggesting 15, 26, and 39 inhibit PDK1 activity. Next
Time (min)
Time (min)
Time (min)
Time (min)
0
10
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50
Time (min)
0
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0.00
-0.05
-0.10
-0.15
0.00
ꢀ0.05
ꢀ0.10
ꢀ0.15
0.00
ꢀ0.08
ꢀ0.16
ꢀ0.24
0.00
-0.05
-0.10
-0.15
0.00
-0.08
-0.16
-0.24
0.0
ꢀ0.20
ꢀ0.5
ꢀ0.32
0.0
0.0
-2.0
0.0
-2.0
-4.0
-8.0
ꢀ4.0
ꢀ8.0
ꢀ1.0
ꢀ1.5
ꢀ2.0
ꢀ2.5
-4.0
-4.0
K
= 290.6 nM
d
Kd = 7.87 uM
Kd = 290.5 nM
G = ꢀ8961 J/mol
Kd = 4.27 ꢀM
-6.0
ꢁG = ꢀ8962 J/mol
-6.0
ꢁ
ꢁ
G = ꢀ6965.2 J/mol
ꢁ
G = ꢀ7325.6 J/mol
-12.0
-16.0
-20.0
Kd = 40.8 ꢀM
-8.0
ꢀ12.0
ꢀ16.0
-8.0
-10.0
-12.0
-10.0
A
D
E
B
C
0.5
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0.00
0.55
1.10
1.65
2.20
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Molar Ratio
Molar Ratio
Molar Ratio
Molar Ratio
Molar Ratio
Figure 3 ITC analysis of the binding mode for 39 and PDK1, raw data for the titration, in which the power output in microcalories per second is measured as
function of time in minutes. A: 39 (100 µM) was titrated into the PDK1 (20 µM) solution; B: ADP (55 µM) was titrated into the PDK1 (20 µM) solution; C:
PDK1 (20 µM) solution and 39 (50 µM) mix together, then ADP (55 µM) was added into the mixture; D: 1 (40 µM) was titrated into the PDK1 (10 µM)
solution; E: PDK1 (10 µM) and 39 (50 µM) mix together, then 1 was titrated into the mixture.
0.65
A
0.7
B
0.72
0.52
0.39
0.26
0.13
0.00
No ATP
0.6
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0.1
0.0
0.60
0.48
0.36
0.24
0.12
0.00
No ATP
Add PDK1
No ATP
PDK1 + DCA (10 mM)
PDK1 + 39 (50 M)
39 (50 M)
Adding ATP
Adding ATP
DCA (10 mM)
ATP + DCA (10 mM)
ATP + 15 (50 ꢀM)
ATP + 26 (50 ꢀM)
ATP + 39 (50 ꢀM)
15 (50 ꢀM)
26 (50 ꢀM)
39 (50 ꢀM)
PDK1 +26 (50 M)
PDK1 +15 (50 M)
0
20
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60
80 100 120 140
0
20
40
60
80
100 120 140
0
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60
80 100 120 140
Time (min)
Time (min)
Time (min)
Figure 4 Compounds 15, 26 and 39 effect on PDC kinetics, OD340 nm values were read at 37 oC. A: 15, 25 and 39 with ATP incubated with PDC at 96ꢀwell
plate; B: 15, 26 and 39 without ATP were incubated in the 96ꢀwell plate; C: PDK1, DCA, 15, 26, 39 and their mixture were added into the PDC solution.
rate (OCR) and extracellular acidification (ECAR) on a Seaꢀ
Compounds 15, 26 and 39 alter the mitochondrial bioener-
horse XFe24 extracellular flux analyzer, and the lactate proꢀ
getics in MCF-7. Inhibition of PDK1 activates PDC, leading
duction was measured by Nova Bioprofile Flex analyzer (Noꢀ
to a switch of pyruvate consumption from lactate production
va Biomedical). As shown in Figure 5, OCR was increased
to oxidative phosphorylation to form acetylꢀCoA in the mitoꢀ
when the cells were treated with 20 µM of 15, 26 and 39. Esꢀ
chondrion. The oxygen consumption and the degree of acidifiꢀ
pecially for 39, the enhancement in OCR is much more signifꢀ
cation will change in cancer cells because of abnormal pyꢀ
icant than that of DCA at 10 mM. ECAR was significantly
ruvate metabolism. The metabolic modulatory effect of 15, 26
decreased with the addition of 15, 26 and 39, which can be
and 39 were evaluated by measuring the oxygen consumption
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explained by the reduction of glycolysis in the cancer cells. As the highest dose for 39, we observed a doseꢀdependent inꢀ
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expected, the proton production rate (PPR) was decreased in a
similar fashion as ECAR. We further compared the ratio of
OCR/ECAR. OCR/ECAR was dramatically increased by 15,
26 and 39. As shown in the Figure 5E, 5F and 5G, except for
crease in OCR and decrease in ECAR and PPR in MCFꢀ7 of
response to 39. In Figure 5H, lactate production was decreased
by 39 at 20 µM after 12 h treatment, which is consistent with
the ECAR change.
C
100
D
B
A
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20
10
0
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90
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30
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***
20
***
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**
*
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***
20
0
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Figure 5 Effect of DCA, 15, 26 and 39 on MCFꢀ7 cell glycolysis. MCFꢀ7 cells (27000/well) were treated with 20 ꢂM and 10 mM DCA, 20 ꢂM 15, 26 and
39 for 12 h. A: OCR was increased with the treatment of DCA, 15, 26 and 39; B: ECAR was significantly decreased by DCA, 15, 26 and 39; C: PPR was
decreased with adding DCA, 15, 26 and 39; as ECAR; D: Ratio of OCR/ECAR was significantly increased by treatment of 15, 26 and 39; E: OCR was
doseꢀdependent increased with the concentration of 39 increases except at the highest dose of 39; F: ECAR was doseꢀdependent decreased with concentraꢀ
tion of 39 increases; G: PPR was decreased with the concentration of 39 increases. H: Lactate formation was decreased by treatment of 15, 26 and 39. * P <
0.05, versus untreated control group.
26 (20 µM)
DCA (10 mM)
15 (20 µM)
39 (20 µM)
DCA (20 µM)
JCꢀ1 green fluorescence
Figure 6 Change in the ꢁψm by JCꢀ1 assay. Treatment of MCFꢀ7 with DCA, 15, 26 and 39 led to the decrease of ꢁψm in the cancer cells. Cells were stained
with JCꢀ1, the green fluorescence represents depolarized mitochondria (Jꢀmonomer), and the red fluorescence represents the hyperpolarized mitochondria
(Jꢀaggregates). The depolarization of ꢁψm is indicated by the increase in the ratio of J monomer / J aggregate. * P < 0.05, versus the group with the treatꢀ
ment of DCA at 20 µM.
for 12 h, followed by staining with JCꢀ1. As shown in the Figꢀ
ure 6, the quantitative analysis of JCꢀ1 stained MCFꢀ7 by 15,
26 and 39 revealed a significant decrease in the red (high
ꢁψm) to green (low ꢁψm) ratio compared with the cells that
treatment of DCA at an equivalent concentration at 20 mΜ.
This was also observed in the JCꢀ1 imaging experiment (see
Figure S5). Compound 39 showed a Jꢀmonomer / Jꢀaggregate
ratio at 1.69 (Figure 6), which was significantly higher than
the ratio given by DCA at 10 mM. Meanwhile, 39 treated cells
displayed a reduction of ꢁψm in doseꢀdependent manner (see
Figure 7). All this suggested that 15, 26 and 39 reverse mitoꢀ
chondria hyperpolarization and oxidative metabolism suppresꢀ
sion in cancer cell. To further validate this result, MCFꢀ7 cells
were stained by tetramethyl rhodamine methyl ester (TMRM),
a fluorescent dye, which accumulate in the hyperpolarized
cells. The 15, 26 and 39 treated MCFꢀ7 cells showed a reducꢀ
Compounds 15, 26 and 39 reverse mitochondria hyperpolari-
zation in cancer cell. Cancer cells exhibit hyperpolarized miꢀ
tochondrial membrane potential (ꢁψm) compared with normal
cells, in which a positive voltage gradient exists across the
27
inner mitochondrial membrane from the outside. Depolariꢀ
zation of cancer cell represents an effective strategy to inhibit
cancer cell growth by introducing apoptosis. 23 Here we invesꢀ
tigated the effect of 15, 26 and 39 on ꢁψm of cancer cells usꢀ
ing
a
cationic
dye,
5,5′,6,6′ꢀtetrachloroꢀ1,1’,3,3’ꢀ
tetraethylbenzimidazolylcarbocyanine iodide (JCꢀ1), which
accumulated in mitochondria accompanied by a fluorescence
emission shift from green to red due to the formation of red
fluorescent Jꢀaggregates. Mitochondria depolarization is indiꢀ
cated by increase in the ratio of green / red fluorescence intenꢀ
sity. In this experiment, MCFꢀ7 cells were treated with DCA
(20 ꢂM and 10 mM), 15 (20 ꢂM), 26 (20 ꢂM) and 39 (20 ꢂM)
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tion of red fluorescent signal (see Figure S6), suggesting 15, micromolar concentrations. Kinetic studies demonstrated that
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26 and 39 led to mitochondrial potential depolarization in
MCFꢀ7 cells. Our results suggest that 15, 26 and 39 provide
apoptotic stimuli for MCFꢀ7 cells through mitochondrial
membrane depolarization, which could lead to the cancer cells
apoptosis.
15, 26 and 39 serve as a PDC activators through inhibition of
PDK1. ITC experiments suggested that 39 binds to ATP bindꢀ
ing pocket instead of the pyruvate binding pocket in PDK1.
The mitochondria bioenergetics measurement revealed that 15,
26 and 39 could serve as a potential modulator to reprogram
the glucose metabolism of cancer cells from lactate formation
to oxidative phosphorylation. Additionally, 15, 26 and 39 were
found to depolarize the mitochondria membrane potential of
cancer cell, which could evolve into apoptotic stimuli leading
to cell death.
CONCLUSION
In this paper, a series of DCA analogues were synthesized and
1
their structures were confirmed by H NMR, 13C NMR and
9
HRMS. Biological assay revealed that three potent compounds,
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namely 15, 26 and 39 inhibited the cancer cells proliferation at
39 (20 µM)
39 (5 µM)
39 (10 µM)
JCꢀ1 green fluorescence
Figure 7 Depolarization of ꢁψm in MCFꢀ7 cells by 39. The cells were treated with 39 for 12 h before measurement.
EXPERIMENTAL SECTION
procedure for compound 15, starting from 4,6ꢀ
dichloropyrimidinꢀ5ꢀamine (1.63 g, 10 mM), 2ꢀdichloroacetyl
chloride (1.06 mL, 11 mmol) and Et3N (1.67 mL, 12 mmol).
The desired compound (1.26 g) was obtained as light yellow
solid. Yield: 47.1 %. Purity: 96.71 %. m.p. 107−108 C. H
NMR (400 MHz, DMSOꢀd6): δ 11.12 (s, 1H), 8.93 (s, 1H),
6.85 (s, 1H) ppm; 13C NMR (100 MHz, DMSOꢀd6): δ 162.59,
159.78, 156.83, 127.92, 66.13; HRMS (ESI) calcd. for
C6H4Cl4N3O [M+H]+, 273.9108; found, 273.9115.
Materials and Methods. All the building blocks were bought
from sigma, JK chemical, Wako and Acros and were used as
received. Solvents were purchased from Anaqua Chemicals
Supply (ACS Grade). Dimethylsulfoxideꢀd6 was bought from
o
1
1
CIL, USA. All the new compounds were characterized by H
NMR, 13C NMR and HRMS. NMR spectra were recorded on a
Bruker AVꢀ400 instrument and reported in ppm downfield
from DMSO (2.50 ppm), and all 13C NMR spectra are reported
in ppm relative to residual DMSO (39.6 ppm). Coupling conꢀ
stants are reported in Hz with multiplicities denoted as s (sinꢀ
glet), d (doublet), t (triplet), q (quartet) and m (multiplet). Reꢀ
actions were monitored by thinꢀlayer chromatography (TLC)
carried out on commercial silica gel plates (GF254) using UV
light as a visualizing agent. High resolution mass spectra
(HRMS) were obtained on a Xevo G2ꢀXS QTof spectrometer.
All of the tested compounds exhibited purities of > 95 % as
determined by peak area integration in reverse phase chromaꢀ
tography through a Cꢀ18 column (Agilent HPLC 1260, USA).
2,2-dichloro-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide
(39). Compound 39 was prepared according to the experiꢀ
mental procedure for compound 15, starting from 4ꢀchloroꢀ3ꢀ
(trifluoromethyl)aniline (0.98 g, 5 mmol), 2ꢀdichloroacetyl
chloride (0.53 mL, 5.5 mmol) and Et3N (0.84 mL, 6 mmol).
The desired compound (0.85 g) was obtained as yellow solid.
Yield: 56.0 %. Purity: 98.64 %. m.p. 96−97 oC. 1H NMR (400
MHz, DMSOꢀd6): δ 11.09 (s, 1H), 8.14 (d, 1H, J = 2.4 Hz),
7.88 (dd, 1H, J = 2.8, 8.8 Hz), 7.70 (d, 1H, J = 8.8 Hz), 6.62
(s, 1H) ppm; 13C NMR (100 MHz, DMSOꢀd6): δ 162.45,
137.22, 132.50, 127.54, 127.23, 126.93, 126.62, 125.64,
124.80, 123.99, 121.27, 118.75, 118.64, 118.58, 67.23. HRMS
(ESI) calcd. for C9H6Cl3F3NO [M+H]+, 305.9467; found,
305.9460.
Apoptosis detection by flow cytometry. MCFꢀ7 cells were
seeded at a density of 2 x 105 cells/mL on each well of a six
well plate and were allowed to grow overnight. Cells were
treated with 10 mM DCA, 20 µM 15, 26 and 39 for 12 h at 37
°C. Additional, time–dependent experiments for studying the
apoptosis induced by 39 were pursed at 5 h, 10 h and 15 h. For
each time point, cells were trypsinized, repeatedly washed
with cold PBS for three times, and centrifuged at 800 RPM for
5 min, and the supernatants were discarded. Cells were resusꢀ
pended in 1 x Annexin binding buffer to ~5×105 cells/mL,
preparing a sufficient volume to have 100 ꢁL per assay. To
100 ꢁL of cell suspension, 10 ꢁL Annexin V and 5 ꢁL PI
working solution were added, and incubated for 15 min at
room temperature. After incubation, 400 ꢁL PBS was added to
Synthesis
of
the
2,2-dichloro-N-(4,6-dichloro-2-
methylpyrimidin-5-yl)acetamide (15). A mixture of 4,6ꢀ
dichloroꢀ2ꢀmethylpyrimidinꢀ5ꢀamine (1.77 g, 10 mmol) and
Et3N (1.67 mL, 12 mmol) were stirred in the DCM (20 mL) at
ice bath. Then 2, 2ꢀdichloroacetyl chloride (1.06 mL, 11
mmol) was added with dropwise. After completion of the reꢀ
action, the mixture was extracted with saturated brine (3 x 30
mL), then the combined organic phase was dried over anhyꢀ
drous sodium sulfate and subsequently the solvent was evapoꢀ
rated under reduced pressure. The residue was purified by
silica gel column chromatography (eluent, petroleum/ethyl
acetate, 10/1, V/V) to obtain 15 (2.20 g) as pale yellow solid.
Yield: 78.5 %. Purity: 95.78 %. m.p. 148−149 C. H NMR
(400 MHz, DMSOꢀd6): δ 10.97 (s, 1H), 6.82 (s, 1H), 2.65 (s,
3H) ppm; 13C NMR (100 MHz, DMSOꢀd6): δ 167.14, 162.69,
159.31, 124.84, 66.71, 25.00; HRMS (ESI) calcd. for
C7H6Cl4N3O [M+H]+, 287.9265; found, 287.9259.
o
1
2,2-dichloro-N-(4,6-dichloropyrimidin-5-yl)acetamide (26).
Compound 26 was prepared according to the experimental
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each sample, and was gently mixed and analyzed immediately sensor cartridge was placed on top of the utility plate, and kept
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on the flow cytometer.
at 37 °C without CO2 for 12 h. MCFꢀ7 cells were cultured in
XF24ꢀwell cell culture microplates (Seahorse Bioscience) at a
density of 2.7 x 104 cells/well in 100 µL growth medium and
incubated for 1 h at 37 °C in 5% CO2 atmosphere. After the
cells were attached, an additional 100 µL growth medium was
added and the cells were incubated for 24 h at 37 °C in 5%
CO2 atmosphere. The cells were treated with DCA (20 ꢂM
and 10 mM), 15 and 26 (20 ꢂM), 39 (5 ꢂM, 10 ꢂM and 20
ꢂM) for 12 h at 37 °C in 5% CO2 atmosphere. After 12 h, the
culture was removed from each well (retaining 50 ꢂL) and the
cells were rinsed two times with 1000 ꢂL of XF stress test
glycolysis optimization medium preꢀwarmed to 37 °C and
finally 1000 ꢂL of optimization medium was added to each
well and the plate was placed at 37 °C without CO2 for 1 h
prior to assay. The OCR, a measure of mitochondrial respiraꢀ
tion, and the ECAR and PPR, markers for glycolysis, were
measured simultaneously for 16 min to establish a baseline
rate.
PDK1 expression and purification. Human PDK1 (aa29ꢀ436)
was cloned from pDONR223ꢀPDK1 (Addgene plasmid
#23804) using primers (tacttccaatccaatgcatcggactcgggctcc and
ttatccacttccaatgttattactaggcactgcggaꢀac) and ligated into pETꢀ
His6SumoꢀTEVꢀLIC (Addgene plasmid #29659). Nꢀterminal
6xHisꢀSUMO tagged recombinant was coꢀexpressed with
chaperonins GroEL and GroES (encoded by Addgene plasmid
#27396) in Escherichia coli BLꢀ21 cells, purified (> 95 %)
through a nickelꢀcharged HiTrap IMAC column and a HiLoad
16/600 Superdex200 column on an AKTA Avant150 chromaꢀ
tography system.
Kinetic study for the PDC. PDK inhibition activity was asꢀ
sessed indirectly by measuring the residual PDC activity after
kinase reaction in an assay, in which the NADH was measured
during the conversion from pyruvate to the acetylꢀCoA. The
assay consisted of the following three steps. (1) Preincubation.
This step was included in the assay because the intrinsic PDK
activity of commercially available PDC was enhanced by up
to 2.7ꢀfold. Pig PDC (sigma, CAS 9014ꢀ20ꢀ4) containing inꢀ
trinsic kinase activity PDKs was incubated for 40 min at 37 °C
in buffer A [40 mM Mops (pH 7.20), 0.5 mM EDTA, 30 mM
KCl, 1.5 mM MgCl2, 0.25 mM acetylꢀCoA, 0.05 mM NADH,
2 mM dithiothreitol, 10 mM NaF] at a concentration of 80
µg/ml. (2) PDK reaction. The PDK reaction was performed in
a total volume of 100 µL at 37 °C, and was initiated by dilutꢀ
ing the PDC mixture 2.2ꢀfold in 1.8 x buffer A containing 55
µM ADP, 100 µM ATP (ATP was omitted in control reactions
representing complete PDK inactivation). After 3 min the kiꢀ
nase reaction was terminated by the addition of 10 µL of stopꢀ
ping buffer (55 mM ADP and 55 mM pyruvate). (3) Residual
PDC activity. The remaining PDC activity was tested in a total
volume of 200 µL at 37 °C by addition of 90 µL of buffer B
[120 mM Tris (pH 7.8), 0.61 mM EDTA, 0.73 mM MgCl2, 2.2
mM thiamine pyrophosphate, 11 mM 2ꢀmercaptoethanol, 2.2
mM NAD+, 2.2 mM pyruvate, 1.1 mM CoA] by measuring the
production of NADH at 340 nm.
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ASSOCIATED CONTENT
Supporting Information: Cell culture, cell cytotoxic assay,
IncyCyte Zoom live cell images, lactate measurement, PDK
kinase activity assay, TMRM assay, JCꢀ1 assay for image,
Figures (Figs. S1ꢀS6), Table S1 and 1H NMR, 13C NMR,
HRMS and HPLC spectra were listed in supporting inforꢀ
mation. This material is available free of charge via the Interꢀ
net at http://pubs.acs.org.
AUTHOR INFORMATION
* Corresponding Author
Prof. Kin Yip Tam (kintam@umac.mo); Tel.: +853 88224988;
fax: +853 88222314.
Author Contributions
# These authors contributed equally to this study.
Notes
The authors declare no competing financial interests.
ITC analysis. The concentration of PDK1 proteins were deꢀ
termined by NanoDrop 2000 and diluted to 10 µM solutions in
the buffer solution containing 50 mM KH2PO4/K2HPO4, pH
7.4, 250 mM KCl, 2 mM MgCl2. Stock solutions of the synꢀ
thesized PDK1 inhibitor 39 and the commercially available
compound 1 were prepared in DMSO (40−100 mM). ATP,
ADP and acetylꢀCoA were dissolved in doubleꢀdistilled water
to the concentrations of 10 mM, 55 mM and 5 mM, respecꢀ
tively. Then the above mentioned solutions were diluted by
buffer solution (50 mM KH2PO4/K2HPO4, pH 7.4, 250 mM
KCl, 2 mM MgCl2) into the desired concentrations containing
1 % DMSO. The corresponding solutions were delivered into
the titration syringe. ITC experiments were accomplished usꢀ
ing a Microcal iTC200 microcalorimeter (GE Healthcare). The
reaction cell contained 300 µL PDK1. Titrations were perꢀ
formed with the injection of 2.5 µL titrant(s) for every increꢀ
ment into the reaction cell which maintained at 25 °C. All of
the ITC data were initially analyzed and the baseline was auꢀ
tomatically determined by the Origin 7, then followed by
curveꢀfitting to oneꢀsite model to obtain binding parameters
ꢁG, ꢁH, ꢁS and n.
ACKNOWLEDGMENT
We thank the financial support from the Science and Technolꢀ
ogy Development Fund, Macao S.A.R (FDCT) (project referꢀ
ence no. 086/2014/A2).
ABBREVIATIONS USED
DCA, dichloroacetate; PDK, Pyruvate dehydrogenase kinase;
PDC, pyruvate dehydrogenase complex; ATP, adenosine triꢀ
phosphate; ADP, adenosine diphosphate; acetylꢀCoA, acetyl
coenzyme A; HIFꢀ1α, hypoxiaꢀinducible factor 1α; OCR, oxꢀ
ygen consumption rate; ECAR, extracellular acidification;
PPR, proton production rate; ꢁψm, mitochondrial membrane
potential; TMRM, tetramethyl rhodamine methyl ester;
NADH, reduced nicotinamide adenine dinucleotide; A549,
human lung adenocarcinoma epithelial cell line; MCFꢀ7,
breast cancer cell line Michigan Cancer Foundationꢀ7.
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