Reverse Chemical Proteomics Identifies an Unanticipated Human Target of the Antimalarial Artesunate

Article abstract of DOI:10.1021/acschembio.8b01004

Artemisinins are the most potent and safe antimalarials available. Despite their clinical potential, no human target for the artemisinins is known. The unbiased interrogation of several human cDNA libraries, displayed on bacteriophage T7, revealed a single human target of artesunate; the intrinsically disordered Bcl-2 antagonist of cell death promoter (BAD). We show that artesunate inhibits the phosphorylation of BAD, thereby promoting the formation of the proapoptotic BAD/Bcl-xL complex and the subsequent intrinsic apoptotic cascade involving cytochrome c release, PARP cleavage, caspase activation, and ultimately cell death. This unanticipated role of BAD as a possible drug target of artesunate points to direct clinical exploitation of artemisinins in the Bcl-xL life/death switch and that artesunate's anticancer activity is, at least in part, independent of reactive oxygen species.

Full text of DOI:10.1021/acschembio.8b01004

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Article
Reverse Chemical Proteomics Identifies an Unanticipated
Human Target of the Antimalarial Artesunate
Michael P. Gotsbacher, Sungmin Cho, Nam Hee Kim, Fei Liu, Ho Jeong Kwon, and Peter Karuso
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b01004 • Publication Date (Web): 06 Mar 2019
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Reverse Chemical Proteomics Identifies an Unanticipated Human
Target of the Antimalarial Artesunate
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1‡†
2‡
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2*
Michael P. Gotsbacher, Sungmin Cho, Nam Hee Kim, Fei Liu, Ho Jeong Kwon, and Peter
Karuso
1*
1Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, Australia.
2Chemical Genomics Global Research Laboratory, Department of Biotechnology, College of Life Science &
Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749
ABSTRACT: Artemisinins are the most potent and safe antimalarials available. Despite their clinical potential, no human
target for the artemisinins is known. The unbiased interrogation of several human cDNA libraries, displayed on
bacteriophage T7, revealed a single human target of artesunate; the intrinsically disordered Bcl-2 antagonist of cell death
promoter (BAD). We show that artesunate inhibits the phosphorylation of BAD, thereby promoting the formation of the
proapoptotic BAD/Bcl-xL complex and the subsequent intrinsic apoptotic cascade involving cytochrome c release, PARP
cleavage, caspase activation and ultimately cell death. This unanticipated role of BAD as a possible drug target of
artesunate points to direct clinical exploitation of artemisinins in the Bcl-xL life/death switch and that artesunate’s
anticancer activity is, at least in part, independent of reactive oxygen species.
The study of weak, but selective, protein–ligand
interactions has always been challenge and is
the infected red blood cell, the endoperoxide bridge of
artemisinin is activated and cleaved through a heme-
a
exemplified by gamma-hydroxybutyric acid, which is a 3
mM agonist of GABA-B receptors but highly selective. Low
dependent, radical mechanism that alkylates parasite
1
18-20
proteins.
This has recently been supported through
affinity effectively hinders the identification of small
molecule protein targets using genome-wide, forward
proteomic methods such as affinity capture MS or affinity
chemical proteomics investigations in which at least 124
parasite proteins were shown to be alkylated by an
artesunate analogue in infected red blood cells.
21
2
chromatography. Even high affinity interactions can be
Notwithstanding this, proposed specific targets include
22
23
problematic to identify in the presence of large amounts
of non-specific interactions. An attractive alternative
approach, particularly suited for weak interactions with low
abundance proteins, is reverse chemical proteomics using
phage display where there is a physical link between the
SERCA, membrane glutathione S-transferase (PfEXP1)
24
or phosphatidylinositol-3-kinase PfPI3K in P. falciparum.
a
Scheme 1. Synthesis of probes and NMR correlations for
probe 3; red = HMBC, green = ROESY, black = COSY,
showing key correlations only.
3-13
genome and proteome.
The rapid life cycle of
bacteriophages allows iterative purification of rare and/or
low abundance proteins from highly complex mixtures
based on relatively weak affinity to a tagged small
molecule. For example, we were able to rapidly isolate a
human protein target for kahalalide F that showed ~50 M
9
K
D
with ribosomal protein S25. Here we report the
identification of the Bcl-2 agonist of cell death (BAD)
promotor as the first human target of the natural product
drug artesunate (1) using T7 phage display.
Artemisinins are sesquiterpenes from the sweet
14, 15
wormwood (Artemisia annua).
They are the most
potent antimalarials available with 500 million
doses/annum prescribed to eradicate Plasmodium
16
falciparum infections but their mode of action is still
17
under debate. It is however widely accepted that inside
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H
O
H
O
extensive primary literature on their anticancer activity, 1’s
human target remains elusive.
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O
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31-34
(
a) NaBH4/MeOH
b) imidazole/DCM
H
H
O
O
O
O
(
O
O
O
O
O
O
2
1
O
COOH
Biotin-PEG-NH2
HN
H
NH
H
H
N
O
O
S
NH2
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O
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(c) EDC/
HOBt/DMF
(
d) EDC/
HOBt/DMF
O
HO
O
HN
H
NH
H
H
N
O
O
N
O
S
H
O
10
Figure 1. BAD is the common protein binding partner of 1,
identified by biopanning of phage displayed cDNA libraries
from various cancer cells. (A) Agarose gel electrophoresis of
phage DNA inserts, amplified by PCR from phage sub-libraries
after biopanning against 5 immobilized on neutravidin-
coated microtiter plates. See Figures S4 and S5 for more
detail. (B) Phage titer after each round of biopanning showing
convergence after rounds 8-10 for the 4 tumor cDNA libraries
and (C) On-phage binding study showing 100-fold higher titer
of the BAD-displaying phage clone for the 1-immobilized
support than the support with immobilized negative control
4
15
4
3
1
O
O
O
2
5
2a
53a
NH
5
3
H
O 6
HN
H
H
O
5
14
5
0
43a
H
19a
10
H
H H
H H
O
16
51
H
H
O
H
H
47
12
52 49
9
46 45 44
N
O
20
N
19 18 17
11
7
H
H
42
4
3
O
21
8
S
H
H H
O
H H 10
H H
O
H H
H
13
3
H
H
O
O
O
O
H
O
O
O
O
HN
OH
5
O
O
N
N
O
H
O
OH
e) EDC/DMAP/DCM
N
N
(
4). The wild-type phage (no insert) does not differentiate the
N
(
O
two supports.
NO2
6
NO2
RESULTS AND DISCUSSION
We began with the synthesis of a biotinylated analog (3)
of 1 from artemisinin (2) containing a long, hydrophilic
a
Reagents and conditions: (a) 2, NaBH
°C, 2 h, 81% yield; (b) 5, succinic anhydride (1.6 equiv),
4
(3.5 equiv)/MeOH, 0–
linker (Biotin-PEG-NH
2
; Scheme 1). Derivatization at C12 is
5
imidazole (0.9 equiv)/DCM, rt 2 h, 91% yield; (c) Biotin-PEG-
NH , 1 (2 equiv), EDC (4 equiv), HOBt (4 equiv)/DMF, 0 °C over
0 min, then rt 18 h, then excess water, 39% yield; (d) Biotin-
PEG-NH , valeric acid (2 equiv), EDC (3 equiv), HOBt (3
equiv)/DMF, 0 °C over 30 min, then rt 18 h, then excess water,
8% yield; (e) NBD-Cl (1.2 equiv)/ACN, -alanine (1.2 equiv)
and NaHCO (3 equiv)/water, 55 °C, 1 h; then remove ACN, pH
to 2 (1 N HCl); solvent removal, then 5 (1 equiv)/DCM, DMAP
1.2 equiv) under N ; then EDC (1.2 equiv), rt 18 h, 42% yield
over two steps.
known to not affect the anticancer activity of
35
artemisinins. The structure, purity, stereochemistry and
stability were verified by HPLC, NMR spectroscopy and
HRMS (Scheme 1; Figures S1–S2). Likewise, a biotinylated
2
3
2
“
blank probe” reagent (4) was synthesized from valeric
acid, along with 6, a fluorescent analog of 1 for imaging
F-ART, Scheme 1; Figure S3). Compound 3 and 4 were
2
3
(
immobilized in separate neutravidin coated microtiter strip
wells. Five human cDNA libraries, expressed in
bacteriophage T7, were panned against 3 coated wells,
after preclearing in 4 wells to remove non-specific binders.
After 9 rounds of biopanning, all four tumor libraries
produced dominant clones but the normal colon library
did not converge (Figure 1A; Figure S4). For all four tumor
libraries, the phage titer increased exponentially after
round 8 (e.g. Figure 1C). Random plaques were picked after
the last round and fingerprinted by Hinf1 digestion (Figure
S5). From sequencing, in all converged tumor libraries, the
dominant clone (Table S4) was the Bcl-2 antagonist of cell
death promoter (BAD). Comparison of the DNA sequences
(
2
Interestingly, artemisinins display polypharmacology, with
profound and selective anticancer activity2
5, 26
and, it has
been suggested, promote apoptosis via mitochondrial
pathways in cancer cell lines.
genes are upregulated upon 1 treatment in cancer cell
lines. These include BUB3, cyclins, CDC25A (proliferation),
27-29
It is known that many
VEGF,
MMP-9,
angiostatin,
thrombospondin-1
27, 30
(
angiogenesis), and Bcl-2, BAX, NF-B (apoptosis).
Despite their clinical potential for treating cancer and the
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produced 100× more phage particles upon elution from 3
showed that for each library the phage selection produced
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6
BAD clones of different lengths, but all were in-frame with
the phage coat protein and covered the full coding
sequence of BAD (Table 1). No other gene was represented
by in-frame clones that appeared in more than one tumor
library.
derivatized wells compared to 4, suggesting a specific
interaction between BAD and 1. As an alternative to
quantitative K measurements, we constructed a peptide
D
library of BAD peptides (31 × 20-mers shifted by 5 aa each
time; Table S5) arrayed onto a glass slide. Staining with 6,
with butylamine-NBD as a negative control (Figure S13),
indicated preferential binding of 6 to a section around
S136, just before the BH3 domain, and to the C-terminal of
the protein (Figure S14).
Table 1. Sequence alignment of BAD clones from colon
tumor (CoT), liver tumor (LiT), lung tumor (LuT) and breast
tumor (BrT) cDNA phage libraries and consensus with
a
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human BAD .
BAD, as a BH3-only, proapoptotic protein, was originally
identified as a partner for Bcl-2 and Bcl-xL in a yeast 2-
hybrid screen and shown to displace BAX to induce
-10
0b
10
20
30
..|....| ....|....| ....|....| ....|....| ....|....|
-------- --SDRAWAQS MFQIPEFEPS EQEDSSSAER GLGPSPAGDG
-------- ----GAWAQS MFQIPEFEPS EQEDSSSAER GLGPSPAGDG
-------- -SRDRAWAQS MFQIPEFEPS EQEDSSSAER GLGPSPAGDG
-------- ----GAWAQS MFQIPEFEPS EQEDSSSAER GLGPSPAGDG
REAAGPGQ GPRDRAWAQS MFQIPEFEPS EQEDSSSAER GLGPSPAGDG
-------- ---------- MFQIPEFEPS EQEDSSSAER GLGPSPAGDG
-------- --rdrAWAQS MFQIPEFEPS EQEDSSSAER GLGPSPAGDG
CoT_B1
LiT_C6
LuT_B7
LuT_G9
BrT_C10
BAD
37
cytochrome c release and caspase-dependent apoptosis.
To validate the dependence of 1’s apoptotic activity on
BAD, cell proliferation assays were performed first using
HeLa (human cervical cancer) cells (Figure S6,), providing
Consensus
40
50
60
70
80
....|....| ....|....| ....|....| ....|....| ....|....|
CoT_B1
LiT_C6
LuT_B7
LuT_G9
BrT_C10
BAD
PSGSGKHHRQ APGLLWDASH QQEQPTSSSH HGGAGAVEIR SRHSSYPAGT
PSGSGKHHRQ APGLLWDASH QQEQPTSSSH HGGAGAVEIR SRHSSYPAGT
PSGSGKHHRQ APGLLWDASH QQEQPTSSSH HGGAGAVEIR SRHSSYPAGT
PSGSGKHHRQ APGLLWDASH QQEQPTSSSH HGGAGAVEIR SRHSSYPAGT
PSGSGKHHRQ APGLLWDASH QQEQPTSSSH HGGAGAVEIR SRHSSYPAGT
PSGSGKHHRQ APGLLWDASH QQEQPTSSSH HGGAGAVEIR SRHSSYPAGT
2
LD50 values of 63, 38 and 12 M (R = 0.94–0.98) for 24, 48
and 72 h 1 treatments, respectively, and established upper
limits on the concentrations of 1 that can be used in live
cells. The apoptotic effect of 1 was also confirmed in HeLa
cells by observation of a dose dependent release of
cytochrome c into the cytosol, PARP cleavage and caspase
activation (Figures S7–S9). Under the microscope, cells
treated with ≥10 M 1 showed typical signs of apoptosis
(Figure S12).
Consensus PSGSGKHHRQ APGLLWDASH QQEQPTSSSH HGGAGAVEIR SRHSSYPAGT
S112
90
100
110
120
130
....|....| ....|....| ....|....| ....|....| ....|....|
EDDEGMGEEP SPFRGRSRSA PPNLWAAQRY GRELRRMSDE FVDSFKKGLP
EDDEGMGEEP SPFRGRSRSA PPNLWAAQRY GRELRRMSDE FVDSFKKGLP
EDDEGMGEEP SPFRGRSRSA PPNLWAAQRY GRELRRMSDE FVDSFKKGLP
EDDEGMGEEP SPFRGRSRSA PPNLWAAQRY GRELRRMSDE FVDSFKKGLP
EDDEGMGEEP SPFRGRSRSA PPNLWAAQRY GRELRRMSDE FVDSFKKGLP
EDDEGMGEEP SPFRGRSRSA PPNLWAAQRY GRELRRMSDE FVDSFKKGLP
CoT_B1
LiT_C6
LuT_B7
LuT_G9
BrT_C10
BAD
Consensus EDDEGMGEEP SPFRGRSRSA PPNLWAAQRY GRELRRMSDE FVDSFKKGLP
S136 S155
140
150
160
....|....| ....|....| ....|....| ....|...
CoT_B1
LiT_C6
LuT_B7
LuT_G9
BrT_C10
BAD
RPKSAGTATQ MRQSSSWTRV FQSWWDRNLG RGSSAPSQ
RPKSAGTATQ MRQSSSWTRV FQSWWDRNLG RGSSAPSQ
RPKSAGTATQ MRQSSSWTRV FQSWWDRNLG RGSSAPSQ
RPKSAGTATQ MRQSSSWTRV FQSWWDRNLG RGSSAPSQ
RPKSAGTATQ MRQSSSWTRV FQSWWDRNLG RGSSAPSQ
RPKSAGTATQ MRQSSSWTRV FQSWWDRNLG RGSSAPSQ
a
The BH3 domain of BAD is highlighted in blue and the
phosphorylated Ser residues in green. Each library converged
on full-length BAD with many different clones varying in N-
and C-terminal sequences but all were in-frame with the coat
protein. The bolded/underlined residues are the consensus
binding domains from the peptide dot blot experiments (see
Table S5, Figures S13–S14).
b
The mouse numbering system is widely used in the literature
and is 36 amino acids longer than human BAD. Thus S99
above is S136 in the mouse sequence and to avoid confusion
the mouse numbering is used throughout.
Several attempts to clone and overexpress BAD as hexaHis
or GST fusions failed due to the intrinsic disorder of the
36
BAD protein. Attempted purification led to extensive
degradation so it was not possible to obtain a pure sample
Figure 2. Validation of the interaction between 1 and BAD
using fluorescently labeled 1 (F-ART, 6). (A) Competition
between 1 (ART) and 6 (F-ART). Panel 1: Treatment of HeLa
cells with Hoechst (blue) and 6 (green). Panel 2: Treatment of
HeLa cells with Hoechst (blue) and 6 (green) in the presence
of 25 M 1. The intensity of 6 was measured using Image J
for reliable K measurements. Consequently, an on-phage
D
binding assay (Figure 1C) was used and showed that the
7
control phage (no insert) produced a background of ~10
phage particles upon elution with SDS in wells derivatized
with 3 or 4. In contrast, BAD-phage (with BAD insert)
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and displayed as a bar graph. (B) Co-localization of 6 (green)
and BAD-Ab (red). Panel 1, 2: Treatment of HeLa cells with
Hoechst (blue) and 10 M 6 (green) and BAD-Ab (red). Panel
1
2
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7
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9
1
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3: showing additional cells. The colocalization of F-ART and
BAD was measured using Image J and expressed as a Pearson
correlation curve.
The binding of ART to BAD was further investigated in
HeLa cells through confocal microscopy. In order to
investigate the binding of ART to BAD in living cells, F-ART
(6) was synthesized and used in a competitive binding
assay. Cells that were pretreated with unlabeled ART (1)
and then stained with 6 (Fig. 2A, panel 2), showing a 60%
reduction in fluorescence, demonstrating that 6 and 1
share the same binding site in the cytosol of HeLa cells
0
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Figure 3. Validation of the interaction between 1 and BAD
using the DARTS assay. (A) Western blotting of BAD, BAX and
actin (loading control) with variable 1 and pronase. (B)-(D)
graphical representation of (B) for BAD (p = 0.00152; one-way
ANOVA), BAX (p = 0.8968) and actin (p = 0.6592), respectively,
run in triplicate. ** Designates p < 0.01 (Students t-test).
(
Figure 2A). BAD and 6 were also found to partially
colocalize in the cytosol of HeLa cells (Figure 2B) and there
2
is a positive correlation (r > 0.75) between the localization
of 6 and BAD, suggesting that 6 and 1 bind to BAD in living
cells. Similar results were obtained for HEK293 cells (Figure
S12). A DARTS assay with HEK293 cell lysates (Figure 3), in
which the binding of a ligand to a protein is detected by
38
either the enhanced or reduced rate of proteolysis,
suggested the binding of 1 to BAD. The proteolysis of BAD,
BAX, and actin in the cell lysate was monitored in the
–
1
presence of increasing pronase (0 to 10 g mL ) and 1 (0
to 400 M) concentrations, following the literature
recommendations to apply 10× higher ligand
concentrations as the upper limit compared to the
effective concentration.38 For actin (Figure 3D) and BAX
(Figure 3C), hydrolysis proceeded in a 1-independent
manner. In contrast, BAD was more readily proteolyzed in
a clear dose-dependent manner (Figure 3B; p = 0.0063),
suggesting that 1 selectively binds to BAD, but not the
related BAX protein or the actin loading control.
Figure 4. Validation of the interaction between 1 and BAD
using siRNA. (A) BAD can be knocked down in live HeLa cells
with 20 nM mixture of 4 siRNAs for BAD (lane 3) relative to
random mixed siRNA (lane 2). (B) Graphical representation of
(
A) carried out in triplicate. (C) Percentage (relative to control)
cell survival of HeLa cells treated with variable 1 for 2472 h.
D) Same as B except HeLa cells were also treated with a 20
The proapoptotic action of 1 was abrogated when BAD
expression was knocked down using a mixture of 4 siRNAs
against BAD (siBAD) (Figure 4). In the presence of 20 nM
siBAD, BAD expression in HeLa cells was reduced by ~60%
compared to random siRNA (Figure 4A and 4B).
Compound 1’s apoptotic effect increased in a time and
dose dependent manner (Figure 4C) that was only slightly
affected by the introduction of a mixture of random siRNA
(Figure 4D). In cells treated with siBAD with 40 M 1, the
suppression of 1’s apoptotic effect was significant (87%
cells surviving with BAD knock-down vs. 65% without;
Figure 4E). The loss of 1’s apoptotic effect with 20 nM
siBAD was most prominent after 72 hours of growth (71%
surviving with BAD knock-down vs 20% without; Figure
(
nM mixture of random siRNA. (E) The same as (D) except cells
were treated with 20 nM siRNA against BAD. *** Designates p
<
0.001 (Students t-test).
The identification of BAD as a target of 1’s apoptotic effect
came as a surprise. Many mechanisms for the anticancer
activity of artemisinins have been proposed with reactive
oxygen species (ROS) generation or mitochondrial induced
apoptosis, involving Bcl-2 family genes, particularly
33, 40, 41
prominent in the literature.
A cell line-dependent
effect of 1 (or its various derivatives) has been reported but
42
no target was identified. More recently, Button et al.
4E). The same effect was observed for all concentrations of
argued that necroptosis is 1’s main mode of action on
43
1
tested again in a dose-dependent manner (p = 0.055). In
Schwannoma cells.
Even more recently, Hamacher
contrast, the apoptotic effect of camptothecin was not
responsive to changes of BAD expression (Figure S11C)
because camptothecin induces apoptosis by targeting
suggests “ferroptosis” in pancreatic cancer cells and that 1
44
did not induce apoptosis or necroptosis. A recent
chemical proteomics study in this journal suggested that
due to heme activation, 1 covalently modified many
39
topoisomerase I.
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proteins (in HeLa cell lysates treated with 10 M hemin) to
achieve its anticancer activity in parallel to its antimalarial
domain (S155; Table 1) renders the binding of BAD to the
1
2
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hydrophobic BH3 binding domain of Bcl-xL unfavorable
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activity. However, this is not consistent with other studies
that discount oxidative damage as necessary for
cytotoxicity of 1 and demonstrate 1-induced apoptosis in
resulting in inhibition of BAD’s proapoptotic function.
The level of S136-pBAD was therefore measured in HeLa
cells 24 h after treating with 1 (0–40 μM) or camptothecin
(positive control; 1–10 M) (Figure 5A). A dose dependent
decrease in phosphorylation on S136 was observed (Figure
5B) with 1, consistent with the peptide array data (Figures
S13–S14). There was also a dose dependent decrease in
the expression levels of Bcl-xL (Figure 5C, 5D). At 20 μM,
BAD phosphorylation was reduced by 60% and Bcl-xL by
20% respectively.
46
a ROS-independent, Bax-mediated manner. The use of a
cell lysate with a high concentration of added hemin is also
not physiologically relevant. The endoperoxide of ART is
47
known to react with free hemin. Our identification of BAD
as a target of 1 opens new questions in ROS-independent
apoptosis and potential avenues for targeted therapies.
Given that Bcl-2/Mcl-1 family members is an emerging
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strategy in development of anticancer therapeutics and
these life/death switches have been suggested as the
The effect of fixed doses of 1 on HeLa cells in the presence
of 0, 0.01, 0.1, 1 or 10 M ABT-737 or camptothecin was
subject to isobolographic analysis (Figure 6) to determine
if 1 would work synergistically or additively with ABT-737,
49
Achilles’ heel of many tumors,
a
BAD-targeted
mechanism of action for 1 in a specific apoptotic pathway
could lead to synergy with other anticancer therapies.50
59
a clinically useful, known BH3-mimic that binds Bcl-xL.
HeLa cells were grown for 24 or 48 hours after treatment
and the LD50 determined in the presence or absence of 1
(
Figure S15). As expected, 1 and camptothecin are
synergistic (Figure 6B) as they target orthogonal pathways
but surprisingly 1 and ABT-737 Camptothecin were also
found to be highly synergistic (Figure 6A), even though
BAD and ABT-737 both putatively bind Bcl-xL to achieve
their apoptotic effect.
Figure 5. 1 reduces the level of S136 phosphorylation on BAD
and Bcl-xL expression levels in HeLa cells. (A) Dose-dependent
reduction in S136 phosphorylation on BAD in HeLa cells on
treatment with 1. (B) Graphical representation of (A) carried
out in triplicate. (C) Dose-dependent reduction of Bcl-xL
Figure 6. 1 is synergistic with ABT-737 and camptothecin. (A)
Isobole analysis (from Figure S15A-C) of the interaction
between 1 and ABT-737 shows a strong synergistic effect at
10 M (square) and 20 M (diamond) 1. (B) Isobole analysis
(from Figure S15d-f) of the interaction between 1 and
camptothecin also shows a synergistic effect at 10 M
(square) and 20 M (diamond).
expression levels upon
representation of (C) carried out in triplicate. * Designates p <
.05, ** designates p < 0.01, *** designates p < 0.001 (Students
1
treatment. (D) Graphical
0
t-test).
BAD can reverse the pro-survival activity of Bcl-xL, but not
that of Bcl-2. Also, in rat ovaries, mutation of BADS136A
results in the reported binding of BAD to Mcl-1 whereas
As the apparent cytotoxicity of 1 is abrogated if BAD is
knocked down in HeLa cells (Figure 4) and the level of
pBAD decreases in a dose dependent way with siBAD
wild-type phosphorylated BAD binds exclusively to 14-3-
51
3.
Regulation of BAD is achieved by cytokine and growth
factor signaling and likely influences numerous aspects of
(
1
Figure 5), this suggests that binding to BAD is required for
’s apoptotic effect and that ROS are not necessarily
48, 52
metabolism, autophagy, and apoptosis.
In a breast
cancer model where PTEN is mutated, BAD is constitutively
phosphorylated and sequestered by 14-3-3, completely
involved in 1’s anticancer activity. Since phosphorylation is
a major regulatory mechanism by which cancer cells inhibit
BAD function to suppress apoptosis, an elegant way to
restore BAD activity, and thus the sensitivity of cells to
apoptotic signaling without affecting the critical roles of
kinases through kinase-based drugs, would be to inhibit
only the phosphorylation of BAD. From our results, it is
possible that this is indeed how 1 achieves its proapoptotic
53
inhibiting its proapoptotic activity. In AML, it was found
that BAD was phosphorylated at S112 and S136 in 41/42
54
clinical samples tested. BAD’s sensitizer activity toward
apoptosis is negatively regulated by kinases (JNK, PKA and
55-57
AKT) through phosphorylation at S112, S136 and S155.
It has been suggested the phosphorylation within the BH3
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activity in HeLa cells. Theoretically, 1’s binding to BAD can is highly synergistic with the BH3 mimetic ABT-737 that
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either; stabilize the Bcl-xL–BAD interaction; reduce the
sequestration of pBAD by 14-3-3 proteins (leaving free
pBAD susceptible to phosphatases); or inhibit the
phosphorylation of BAD, which favors the formation of the
proapoptotic Bcl-xL–BAD complex. While our observations
binds Bcl-xL. Biophysical characterization of the interaction
between ART and BAD was made difficult because BAD is
an intrinsically disordered protein. Identifying and
quantifying such weak and dynamic interactions is
problematic and biophysical evidence usually more
ambiguous than for globular proteins. However, these
atypical and often weak interactions, refractory to in vivo
characterization, can be very meaningful in higher-order
signalling assemblies and regulation that is only now being
(
Figure 1C, Figure S14) support the last mode of action, the
first two possibilities remain to be refuted.
The combined effects of 1 and ABT-737, a BH3 mimic that
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binds to Bcl-2, Bcl-xL and Bcl-w, but not Mcl-1,
64
appreciated. However, overall, our data suggests a
surprisingly revealed a highly synergistic action (Figure 6).
HeLa cells are resistant to ABT-737 (LD50 > 200 M),
because of high levels of Mcl-1. Our results suggest that
the dephosphorylation (or inhibition of phosphorylation)
of BAD by 1 results in a marked increase in sensitivity of
HeLa cells to ABT-737 (LD50 16 M) in the presence of 10
targeted mechanism of action for 1 for immediate clinical
50
exploitation.
METHODS
See Supporting Information.
M 1 and 1.2 M with 20 M 1. This synergy suggests that
1
may do more than just promote the binding of BAD to
ASSOCIATED CONTENT
Bcl-xL, and we anticipate that this target identification will
enable the future exploration of many of these interesting
possibilities in the clinic. Intrinsically disordered proteins
Supporting Information. Detailed synthetic procedures and
probe stability, detailed target identification and validation
procedures and synergistic activity data. This material is
available free of charge via the internet at http://pubs.acs.org
at DOI:.
(
such as BAD) have the potential to bind to multiple
partners depending on their conformation and post-
translational modifications. Future clinical investigations of
how 1 may sensitize tumors to other genotoxic agents will
shed more light on the tumor selectivity of 1 and inform
clinical repositioning of this fascinating natural product.
Indeed, it is known that 1 synergistically induces apoptosis
of HeLa cells after IR but not SiHa cells
dihydroartemisinin (5) synergistically induces apoptosis in
OVCAR-3 and A2780 (but not IOSE144) ovarian cancer
cells treated with carboplatin. Similarly, A2780, HO8910
AUTHOR INFORMATION
Corresponding Author
*
peter.karuso@mq.edu.au and kwonhj@yonsei.ac.kr
Present Addresses
Current address: School of Medical Sciences
Pharmacology), The University of Sydney, Sydney NSW
006.
60
and
†
(
2
61
and HEY ovarian cancer cells responded to 1 in a dose
dependent manner and are synergistic with carboplatin
but SKOV3 cells were totally unresponsive to 1. 1 has also
been reported to sensitize breast cancer cells to the
Author Contributions
62
‡These authors contributed equally.
Funding Sources
63
chemotherapeutic agent epirubicin.
No competing financial interests have been declared.
This work was supported by ARC grant DP130103281 to
PK and HJK, NRF grant 2015K1A1A2028365,
Follow-up studies will include further characterization of
the interaction between ART and BAD using other
biophysical techniques and phosphoproteomics. The
synergy between ART and AZD-59912 and S63845
(inhibitors of Mcl-1) and the interaction of ART with other
BH3-only proteins will help to further characterize the
cellular mechanism of ART that would not be obvious
without first identifying BAD as a target of ART.
2015M3A9C4076321 and BK21plus to HJK.
ABBREVIATIONS
BAD, Bcl-2 Antagonist of cell Death; pBAD, S136
phosphorylated BAD; siBAD, a mixture of 4 specific small
interfering RNAs against BAD; ART, artesunate; F-ART,
fluorescent ART.
In summary, phage display is an underutilized but powerful
technique for the genome wide, unbiased, reverse
chemical proteomics identification of potential protein
targets of small molecules. We have identified the Bcl-2
associate death promoter (BAD) as a possible human
target of artesunate (1) in several human cancer
proteomes displayed on bacteriophage T7 and shown that
a possible mode of action is to interfere with BAD
phosphorylation at S136. This BAD-targeting activity of 1
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The antimalarial artesunate targets BAD in the Bcl-xL proapoptotic pathway in humans
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