Natural and Semisynthetic Chalcones as Dual FLT3 and Microtubule Polymerization Inhibitors

Article abstract of DOI:10.1021/acs.jnatprod.0c00699

Activating mutations in FLT3 receptor tyrosine kinase are found in a third of acute myeloid leukemia (AML) patients and are associated with disease relapse and a poor prognosis. The majority of these mutations are internal tandem duplications (ITDs) in the juxtamembrane domain of FLT3, which have been validated as a therapeutic target. The clinical success of selective inhibitors targeting oncogenic FLT3, however, has been limited due to the acquisition of drug resistance. Herein the identification of a dual FLT3/microtubule polymerization inhibitor, chalcone 4 (2′-allyloxy-4,4′-dimethoxychalcone), is reported through screening of 15 related chalcones for differential antiproliferative activity in leukemia cell lines dependent on FLT3-ITD (MV-4-11) or BCR-ABL (K562) oncogenes and by subsequent screening for mitotic inducers in the HCT116 cell line. Three natural chalcones (1-3) were found to be differentially more potent toward the MV-4-11 (FLT3-ITD) cell line compared to the K562 (BCR-ABL) cell line. Notably, the new semisynthetic chalcone 4, which is a 2′-O-allyl analogue of the natural chalcone 3, was found to be more potent toward the FLT3-ITD+ cell line and inhibited FLT3 signaling in FLT3-dependent cells. An in vitro kinase assay confirmed that chalcone 4 directly inhibited FLT3. Moreover, chalcone 4 induced mitotic arrest in these cells and inhibited tubulin polymerization in both cellular and biochemical assays. Treatment of MV-4-11 cells with this inhibitor for 24 and 48 h resulted in apoptotic cell death. Finally, chalcone 4 was able to overcome TKD mutation-mediated acquired resistance to FLT3 inhibitors in a MOLM-13 cell line expressing FLT3-ITD with the D835Y mutation. Chalcone 4 is, therefore, a promising lead for the discovery of dual-target FLT3 inhibitors.

Full text of DOI:10.1021/acs.jnatprod.0c00699

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Article
Natural and Semisynthetic Chalcones as Dual FLT3 and Microtubule
Polymerization Inhibitors
Haleema Sadia Malik, Aishah Bilal, Rahim Ullah, Maheen Iqbal, Sardraz Khan, Ishtiaq Ahmed,
Karsten Krohn, Rahman Shah Zaib Saleem, Hidayat Hussain,* and Amir Faisal*
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ABSTRACT: Activating mutations in FLT3 receptor tyrosine kinase
are found in a third of acute myeloid leukemia (AML) patients and
are associated with disease relapse and a poor prognosis. The majority
of these mutations are internal tandem duplications (ITDs) in the
juxtamembrane domain of FLT3, which have been validated as a
therapeutic target. The clinical success of selective inhibitors targeting
oncogenic FLT3, however, has been limited due to the acquisition of
drug resistance. Herein the identification of a dual FLT3/microtubule
polymerization inhibitor, chalcone 4 (2′-allyloxy-4,4′-dimethoxychal-
cone), is reported through screening of 15 related chalcones for
differential antiproliferative activity in leukemia cell lines dependent
on FLT3-ITD (MV-4-11) or BCR-ABL (K562) oncogenes and by
subsequent screening for mitotic inducers in the HCT116 cell line.
Three natural chalcones (1−3) were found to be differentially more potent toward the MV-4-11 (FLT3-ITD) cell line compared to
the K562 (BCR-ABL) cell line. Notably, the new semisynthetic chalcone 4, which is a 2′-O-allyl analogue of the natural chalcone 3,
was found to be more potent toward the FLT3-ITD+ cell line and inhibited FLT3 signaling in FLT3-dependent cells. An in vitro
kinase assay confirmed that chalcone 4 directly inhibited FLT3. Moreover, chalcone 4 induced mitotic arrest in these cells and
inhibited tubulin polymerization in both cellular and biochemical assays. Treatment of MV-4-11 cells with this inhibitor for 24 and
48 h resulted in apoptotic cell death. Finally, chalcone 4 was able to overcome TKD mutation-mediated acquired resistance to FLT3
inhibitors in a MOLM-13 cell line expressing FLT3-ITD with the D835Y mutation. Chalcone 4 is, therefore, a promising lead for the
discovery of dual-target FLT3 inhibitors.
cute myeloid leukemia (AML) results from interrupted
differentiation and maturation of hematopoietic progen-
imately 3−400 base pairs in length and act as gain of function
mutations by promoting receptor dimerization in the absence
of the ligand, thereby constitutively activating the receptor.8
Point mutations in the TKD are less frequent when compared
to the ITD mutations and also result in ligand-independent
activation of the FLT3 receptor.9 Both of these activating
mutations are associated with disease relapse and poor
prognosis.
Activated FLT3 promotes cell proliferation and survival
through multiple downstream signaling pathways, including
phosphatidylinositol 3-kinase (PI3K)/AKT, signal transducer
and activator of transcription 5 (STAT5), and extracellular
signal-regulated kinase (ERK).10,11 Many small-molecule FLT3
inhibitors have been developed and evaluated in clinical trials,
A
itor cells, leading to uncontrolled proliferation and accumu-
lation of myeloid blasts. It is the most frequent form of myeloid
leukemia, and more than a quarter of a million adults are
diagnosed with this disease each year.1,2 Mutations in the
nucleophosmin 1 (NPM1), FMS-like tyrosine kinase 3
(FLT3), and DNA methyltransferase 3A (DNMT3A) genes
are the most frequent mutations associated with AML. FLT3
belongs to the class III receptor tyrosine kinase (RTK) family,
which is characterized by structural features such as
extracellular immunoglobulin-like domains, a transmembrane
part, a juxtamembrane (JM) domain, and an intracellular
tyrosine kinase domain (TKD) split into two.3,4 The JM
domain plays an inhibitory role and prevents the activation
loop from acquiring the active conformation in the absence of
the ligand. Binding of the FLT3-ligand (FL) to its receptor
causes it to undergo a conformational change leading to its
homodimerization and subsequent activation. The internal
tandem duplications (ITDs) in the JM domain are the most
frequent mutations in FLT3.5−7 The ITDs are in-frame
insertion of duplicated sequences, which comprise approx-
Received: June 23, 2020
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Scheme 1. Preparation of Chalcones 1−4 and Structures of Chalcones 5−15
including sorafenib,12 lestaurtinib,13 and quizartinib.14 Two
FLT3 inhibitors, midostaurin and gilteritinib, have also been
approved for the treatment of FLT3-ITD+ AML patients.15,16
The efficacy of many of these inhibitors as a single agent,
however, has been limited by transient responses and the
development of acquired resistance.17,18 Acquisition of
secondary mutations in the TKD domain represents the
predominant drug resistance mechanism,19 and mutations at
F691 and D835 residues in the TKD domain have been
identified in relapsed patients undergoing treatment with FLT3
inhibitors.20,21
Overcoming TKD-mediated drug resistance thus represents
a major challenge for the treatment of FLT3-ITD-driven
tumors.22 Discovery of drugs that can inhibit FLT3-TKD
mutants, administration of drugs in combination and
polypharmacology for engagement of targets in addition to
FLT3 have been employed to overcome primary and acquired
resistance to FLT3 inhibitors.23−25 Several dual FLT3
inhibitors have been shown to overcome TKD-mediated
drug resistance. Examples of dual FLT3 inhibitors include
AMG 925, which inhibits FLT3/CDK4, and tandutinib, which
targets FLT3/PDGFR.23 Similarly, inhibitors that target
Aurora kinases in addition to FLT3 can overcome TKD-
mediated acquired resistance to FLT3 inhibitors.26,27
itors have been shown to target microtubule dynamics in
addition to their primary targets.30 Tivantinib is a c-Met
inhibitor, which, for example, was initially developed to inhibit
the proliferation of cells expressing wild-type or mutant forms
of c-Met.31 It was, however, found to inhibit the proliferation
of cells without c-Met expression owing to its ability to bind to
microtubules at the colchicine-binding site.32,33 Similarly, 3-
substituted 7-phenylpyrrolo[3,2-f ]quinolin-9(6H)-one was
initially discovered as a microtubule polymerization inhibitor
but was later found to inhibit multiple kinases, including
FLT3.34
Chalcones are a flavonoid subgroup that are distributed
widely in fruits, vegetables, teas, and plants.35 Chalcones have
been reported to exhibit numerous biological properties such
as anti-inflammatory, antimicrobial, antifungal, antimalarial,
antidiabetic, anti-HIV, antiprotozoal, antioxidant, and cytotoxic
activities.35,36 In addition, several chalcone-based molecules
have been authorized for clinical practice.35 Herein are
presented the identification and characterization of natural
chalcones (1−3) and in particular the semisynthetic chalcone
4 as a dual FLT3/microtubule polymerization inhibitor.
Chalcone 4 has been found to be preferentially more active
in FLT3-ITD-expressing cells and inhibits FLT3 directly in a
biochemical assay. It was also determined that chalcone 4
induces mitotic arrest and inhibits microtubule polymerization
in vitro. The dual inhibition of FLT3 and microtubules causes
apoptotic cell death and is able to overcome D835Y-mediated
resistance to FLT3 inhibitors.
Microtubules are dynamic structures that are critical for the
completion of mitosis and the division of cells, in addition to
their role in cell signaling, vesicular transport, and cytoskeleton
dynamics.28 The highly dynamic nature of spindle micro-
tubules has been utilized successfully to target rapidly
proliferating cancer cells with drugs that interfere with
microtubule dynamics.29 Many small-molecule kinase inhib-
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Table 1. GI50 Values of Chalcones 1−4 in Three AML Cell Lines
a
GI50 SEM (μM)
compound no.
MOLM-13
MV-4-11
THP-1
1
2
3
4
>10
10 1.91 (n = 4)
>10
0.20 0.096 (n = 3)
>10
>10
>10
>10
>100
>100
0.24 0.015 (n = 3)
5.4 1.615 (n = 4)
a
Data represent mean values standard error of mean (SEM) for at least two independent biological replicates.
Figure 1. Chalcone 4 is an FLT3 inhibitor. (A) Inhibition of FLT3 downstream signaling by chalcone 4 in MV-4-11 cells. Cells were treated with
indicated concentrations of chalcone 4 for 2 h, followed by analysis of Stat5 and Erk phosphorylation through immunoblotting. Levels of total Stat5
and total Erk were used as loading controls. (B) Docking of chalcone 4 with FLT3. (C) Chalcone 4 directly inhibits FLT3. IC50 of chalcone 4 was
determined in an FLT3 kinase assay at 10 μM ATP concentration.
(2),39 and 4,4′-dimethoxy-2′-hydroxychalcone (3)40,41
RESULTS AND DISCUSSION
■
(Scheme 1), were found to be differentially more potent
Chemistry. The natural chalcones 1−3 and the semi-
synthetic new analogue 4 (Scheme 1) were prepared using the
Claisen−Schmidt condensation protocol. Furthermore, the
new chalcone 4 was prepared by treating natural chalcone 3
toward the MV-4-11 (FLT3-ITD) cell line compared to the
K562 (BCR-ABL) cell line at both concentrations [Figure S1,
Supporting Information (pink outline)]. It is noteworthy that
the new synthesized chalcone 4, which is the 2′-allyloxy
analogue of the natural chalcone 3, inhibited the growth of
both cell lines by >95% at the two concentrations tested (25
and 50 μM; Figure S1, Supporting Information). However, the
natural chalcone 535 and the semisynthetic chalcones 6−1536
were not effective toward the two cell lines used.
The GI50 values of chalcones 1−4 were next determined in
two AML cell lines with oncogenic FLT3-ITD mutations
(MV-4-11 and MOLM-13) and one AML cell line with wild-
type (WT) FLT3 (THP-1). As shown in Table 1, chalcones
1−3 had GI50 values ≥ 10 μM in all three cell lines with
with allyl bromide in the presence of KF-Al2O3 (Scheme 1).
The chalcone 5 (4-methoxylonchocarpin) was isolated from
the plant Dorstenia poinsettifolia.37 The chemistry of the
synthetic chalcones 6−15 was already published by our
group.36
Identification of Chalcones as FLT3 Inhibitors.
Initially, all the chalcones obtained were screened in leukemia
cell lines expressing FLT3-ITD (MV-4-11) or BCR-ABL
(K562) at 25 and 50 μM concentrations in three-day MTS
proliferation assays. The three natural chalcones, viz., 2′-
hydroxychalcone (1),38 2′-hydroxy-3,4,4′-trimethoxychalcone
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Figure 2. Chalcone 4 induces mitotic arrest and inhibits tubulin polymerization in cells. (A) Dot-blot assay for histone H3 phosphorylation at S10
following treatment with 25 μM for 8 h. DMSO and paclitaxel were used as a solvent and positive control, respectively (B) Chalcone 4 induces
histone H3 phosphorylation at S10 in HCT116 and MV-4-11 cells. Cells were treated with the indicated concentrations of chalcone 4 for 8 h and
analyzed for histone H3 phosphorylation using Western blotting. (C) MV-4-11 cells were treated with the indicated concentrations of chalcone 4
and analyzed for histone H3 phosphorylation at S10 through FACS following propidium iodide staining. (D) Chalcone 4 inhibits microtubule
repolymerization in cells. Cells treated with DMSO, chalcone 4, or nocodazole were incubated on ice for 30 min followed by incubation at 37 °C
for 10 min. Microtubules were analyzed through immunofluorescence using anti-tubulin antibodies.
relative selectivity of chalcones 2 and 3 toward AML cell lines
containing FLT3-ITD mutation (GI50 > 10 μM) compared to
the THP-1 cell line, which contains WT FLT3 (GI50 > 100
μM). On the other hand, the 2′-allyloxy derivative of natural
chalcone 3, viz., chalcone 4, was the most selective and potent
of the four chalcones toward FLT3-ITD cell lines, with GI50
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Figure 3. Chalcone 4 is a microtubule polymerization inhibitor. (A) Chalcone 4 inhibits tubulin polymerization in a biochemical assay. The
polymerization assay was performed with purified tubulin in the presence of DMSO, chalcone 4 (5 and 25 μM), and nocodazole (10 μM). (B)
Docking of chalcone 4 in the colchicine-binding site of tubulin. (C) Overlap of chalcone 4 (red) and colchicine (orange) in the colchicine-binding
cavity of tubulin D. Colchicine displacement by chalcone 4. Increasing concentrations of chalcone 4 were incubated with tubulin in the presence of
20 μM colchicine, and the fluorescence intensity was measured on a plate reader. The graph represents fluorescence intensity relative to the DMSO
control.
values ranging from 0.20 to 0.24 μM (Table 1). Interestingly,
the allyloxy group at C-2′ of chalcone 4 dramatically enhanced
the activity toward MOLM-13 (>130-fold), MV-4-11 (>160-
fold), and THP-1 (>45-fold) cells, when compared with the
activity of the parent natural product 3.
through pi-stacking. The binding mode also placed the
carbonyl group of the chalcone 4 in a position to have a
conventional hydrogen bond with Lys644. Chalcone 4 binds to
the same region as tyrosine kinase inhibitor (TKI) quizartinib
through interactions with the “gatekeeper” residue Phe691 and
the DFG motif residue Phe830. Thus, it seems plausible to
assume that chalcone 4 may act by controlling access to an
allosteric pocket adjacent to the ATP-binding site (Figure 1B).
The docking predictions were confirmed through direct
inhibition of FLT3 by chalcone 4 in a biochemical kinase
assay (Figure 1C). Chalcone 4 inhibited FLT3 with an IC50
value of 5.7 μM in the presence of 10 μM ATP. Two other
compounds, chalcone 1 and chalcone 2, were also evaluated in
this kinase assay. Chalcone 2 inhibited FLT3 with an IC50
value of 13.8 μM, while chalcone 1 inhibited FLT3 by 39.5% at
a 100 μM concentration (Figure S3, Supporting Information).
The biochemical IC50 value of chalcone 4, however, was
significantly higher than its GI50 in FLT3-ITD cells. Although
chalcone 4 inhibited FLT3 signaling more potently in MV-4-
11 cells than FLT3 activity in a biochemical assay (IC50 value
of 5.7 μM; Figure 1A), the GI50 value of 244 nM in MV-4-11
cells suggested inhibition of a possible additional target.
Chalcone 4 Inhibits Microtubule Polymerization. In
addition to the screening in leukemia cell lines, screening of the
chalcones for mitotic induction (histone H3 phosphorylation
at S10) in a colon carcinoma cell line (HCT116) using a dot-
The selectivity of these chalcones, particularly of chalcone 4
(>20 fold selective), for the FLT3-ITD cells compared to
FLT3-WT cells, indicated that they might interfere with
oncogenic FLT3 signaling in FLT3-ITD cells. It was therefore
determined whether these compounds could inhibit FLT3
signaling in these cells by measuring the phosphorylation of
Erk, Stat5, or both. Treatment of MV-4-11 cells with all four
compounds resulted in reduced phosphorylation of Stat5 and/
or Erk, two key downstream targets of FLT3 (Figure 1 and
Figure S2, Supporting Information). In line with its potency
and selectivity in FLT3-ITD cell lines, chalcone 4 was more
robust at inhibiting the phosphorylation of both Stat5 and Erk
(Figure 1A).
Inhibition of two signaling proteins downstream of FLT3-
ITD indicated that chalcone 4 might inhibit FLT3, a common
activator upstream of both these proteins. Thus, docking of
chalcone 4 with FLT3 using AutoDock Vina was investigated,
and the computational outcome was analyzed for the plausible
binding modes using Discovery Studio Visualizer. Chalcone 4
was found to interact with Val624 through a pi−sigma
interaction and, more importantly, with Phe691 and Phe830
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Figure 4. Chalcone 4 induces apoptosis in MV-4-11 cells. (A) Chalcone 4 causes caspase-3 and PARP cleavage. Cells were treated with the
indicated concentration of the inhibitor for 24 h, and caspase-3/PARP cleavage was analyzed through Western blotting. (B) Chalcone 4 increase
annexin V binding and propidium iodide uptake. Cells were treated with the indicated concentrations of chalcone 4 for 24 or 48 h and fixed and
stained with FITC-annexin V/PI. Stained cells were then analyzed through FACS.
blot assay was performed. As shown in Figure 2A, chalcone 4
was a robust inducer of histone H3 phosphorylation at S10
following an 8 h treatment at a 25 μM concentration.
In order to confirm that chalcone 4 indeed binds to and
inhibits microtubule polymerization, its effect in a biochemical
assay using purified tubulin was determined. As shown in
Figure 3A, chalcone 4 inhibited microtubule polymerization in
a dose-dependent manner (at 5 and 25 μM concentrations).
DMSO was used as solvent control, while nocodazole (10 μM)
was used as a positive control for depolymerization (Figure
3A). Docking studies of chalcone 4 with tubulin subsequently
were performed. Tubulin has three binding sites, viz., the
paclitaxel-binding site, the vinca alkaloid-binding site, and the
colchicine-binding site. Polymerization inhibitors like colchi-
cine bind at the colchicine-binding site. In agreement with the
bioassays used, the docking predicted that chalcone 4 binds to
tubulin at the colchicine-binding site, with one of the aromatic
rings occupying the same region as occupied by the
trimethoxyphenyl ring of colchicine (Figure 3B and C).
Chalcone 4 undergoes two hydrogen bond interactions with
the tubulin at the Leu255 and Cys241 residues. The phenyl
ring adjacent to the carbonyl group shows pi−sigma
interaction with Leu248, while the other aromatic ring
interacts with the Ala180 and Lys 254 residues. The docking
Treatment of HCT116 cells or MV-4-11 cells with different
concentrations of chalcone 4 for 8 h caused a strong increase in
histone H3 phosphorylation at S10 even at the lowest tested
concentration of 0.78 μM (Figure 2B). Induction of mitotic
arrest following treatment with chalcone 4 was also confirmed
through FACS analysis following treatment of MV-4-11 cells
with different concentrations of chalcone 4 for 24 h (Figure
2C). Induction of mitotic arrest is often a consequence of
interference with microtubule dynamics. Therefore, the effect
of chalcone 4 on microtubule polymerization was determined
through an immunofluorescence-based assay in A549 cells.
Treatment of cells with 5 μM chalcone 4 resulted in complete
inhibition of microtubule repolymerization following cold-
induced microtubule depolymerization (Figure 2D). Cells
treated with the solvent control DMSO were able to
completely repolymerize their microtubules following incuba-
tion at 37 °C, while microtubules in nocodazole-treated cells
remained depolymerized (Figure 2D).
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Figure 5. Chalcone 4 overcomes TKD-mediated resistance to FLT3 inhibitors. (A) GI50 graphs for chalcone 4 and MLN518 in MOLM-13 and
MOLM-13-Res cell lines in a three-day MTS proliferation assay. (B) Table summarizing the GI50 values and fold resistance of two inhibitors using
the MOLM-13 and MOLM-13-Res cell lines.
prediction for association with the colchicine-binding site was
confirmed through a colchicine displacement assay. Chalcone
4 was able to displace colchicine significantly at all three
concentrations used (Figure 3D) with 75%, 80%, and 87%
colchicine displacement at 12.5, 25, and 50 μM, respectively.
The positive control, nocodazole, caused 93.5% displacement
at a 50 μM concentration in the same assay (Figure 3D).
Chalcone 4 Induces Apoptosis in MV-4-11 Cells. Next,
the ability of chalcone 4 to induce apoptosis in MV-4-11 cells
was determined through analysis of activation of caspase-3, an
executioner caspase, or expression of an apoptotic marker,
phosphatidylserine, on the cell surface. Treatment of MV-4-11
cells with different concentrations of chalcone 4 (0.313, 0.625,
1.25, 2.5, and 5 μM) for 24 h resulted in increased cleavage of
caspase 3, indicating its activation (Figure 4A). Cleavage of
caspase-3 was accompanied by cleavage of poly(ADP-ribose)
polymerase (PARP), a substrate of caspase-3. Similarly,
treatment of MV-4-11 cells with different concentrations of
chalcone 4 for 24 and 48 h induced annexin V binding and
propidium iodide retention, indicating early and late apoptotic
cell death (Figure 4B).
Chalcone 4 Overcomes FLT3-TKD-Mediated Resist-
ance. Considering that chalcone 4 could inhibit tubulin
polymerization in addition to inhibiting FLT3, its ability to
overcome D835Y-mediated resistance to FLT3 inhibitors was
evaluated using the MOLM-13 and MOLM-13-Res (with
D835Y mutation) cell lines. Parental MOLM-13 cells were
sensitive to MLN518 (tandutinib) with a GI50 value of 52 nM
(Figure 5). MOLM-13-Res cells, generated through incubation
with increasing concentrations of MLN518,26 were, however,
resistant to MLN518 with a GI50 value of 1.9 μM (36-fold
resistant). Chalcone 4, on the contrary, was equally potent in
MOLM-13 and MOLM-13-Res cell lines with a GI50 value of
0.22 μM in both the cell lines (Figure 5). This shows that
chalcone 4 can inhibit the growth of MOLM-13 cells
irrespective of the TKD mutation status, probably because of
its dual inhibition of FLT3 and microtubule polymerization.
Privileged scaffolds have a wide range of applications in
medicinal chemistry for drug discovery. The flavonoid subtype
chalcone is considered as a privileged scaffold and is present in
numerous natural products.35 Both natural and synthetic
chalcones have illustrated many interesting biological activities
with potential clinical applications for various diseases,
including cancer.35,36,42 Some chalcone derivatives, for
example, have demonstrated promising activity toward T-cell
acute lymphoblastic leukemia (T-ALL)43,44 and CML45 cell
lines.
Dependence of cancer cells on the activity of a single
oncogene (oncogene addiction) makes them sensitive to
inhibition of that oncogene or its downstream signaling
pathway.46 Chronic myeloid leukemia (CML) cells, for
example, are dependent on the BCR-ABL oncogene, and
their dependence on the constitutive activity of ABL kinase has
been successfully exploited for the treatment of CML.47
Similarly, the reliance of a subset of AML cells on constitutive
activation of FLT3 for proliferation and survival makes them
sensitive to FLT3 inhibition, as discussed above.22 The
oncogenic dependence of MV-4-11 and K-562 cell lines to
FLT3-ITD and BCR-ABL, respectively, was exploited for
screening the chalcone library. Differential antiproliferative
activity of chalcones in one or the other cell line was used as an
indicator of interference with the signaling through FLT3-ITD
or BCR-ABL oncogenes. As a consequence, chalcone 4 was
identified as being more active in FLT3-ITD cells, where it
inhibited the phosphorylation of two of the downstream
targets of activated FLT3, STAT5 and ERK.
Actively dividing cancer cells are known to elude apoptosis,
which is why diverse anticancer drugs work by ultimately
promoting apoptotic cell death through related mechanisms.48
Caspase 3, a cysteine protease, is an important factor in
executing the apoptotic process. Once activated through
cleavage, it proteolytically cleaves downstream substrates
such as PARP.49 Mitotic arrest induced by microtubule-
targeting agents activates the spindle assembly checkpoint and
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(1H, s, OH); ESIMS m/z 247.1 [M + Na]+, C15H12NaO2; anal. calcd
for C15H12O2, C 80.34, H 5.39; found: C, 80.27, H 5.33.
eventually leads to apoptotic cell death.50 FLT3 inhibitors also
prevent proliferation by triggering cell cycle arrest and
inducing apoptosis.6 Chalcone 4, which inhibits both micro-
tubule polymerization and oncogenic FLT3 signaling in MV-4-
11 cells, induced apoptotic cell death in these cells as
determined by various methods including increased caspase
3 and PARP cleavage as well as annexin V binding and
propidium iodide uptake.
3,4,4′-Trimethoxy-2′-hydroxychalcone (2). Chalcone 2 was
prepared according to a general procedure, and spectroscopic data
were in good agreement with previously published data:58 mp 159 °C;
1H NMR (500 MHz, CDCl3) δ 3.88 (3H, s), 3.96 (3H, s), 3.98 (3H,
s), 6.50 (2H, m), 6.92 (1H, d, J = 8.4 Hz), 7.16 (1H, bs), 7.26 (1H, d,
J = 1.6 Hz), 7.45 (1H, d, J = 15.6 Hz), 7.85 (1H, s), 7.87 (1H, d, J =
15.6 Hz), 13.54 (1H, s, OH-2′),; ESIMS m/z 336.9 [M + Na]+,
C18H18NaO5; anal. calcd for C18H18O5, C 68.78, H 5.77; found: C
68.74, H 5.71.
4,4′-Dimethoxy-2′-hydroxychalcone (3). Chalcone 3 was
prepared according to a general procedure, and spectroscopic data
were in good agreement with previously published data:59 mp 91−93
°C; 1H NMR (500 MHz, CDCl3) δ 3.86 (3H, s, OCH3), 3.87 (3H, s,
OCH3), 6.49−6.47 (2H, m, H-5′, H-3′), 6.95 (1H, d, J = 8.8 Hz, H-
5), 7.48 (1H, d, J = 15.9 Hz, H-α), 7.62 (2H, d, J = 8.8 Hz, H-2, H-6),
7.85 (1H, d, J = 8.8, H-6′), 7.88 (1H, d, J = 15.9 Hz, H-β), 13.54 (1H,
s, OH-2′); ESIMS m/z 307.3 [M + Na]+, C17H16NaO4; anal. calcd for
C17H16O4, C 71.82, H 5.67; found: C 71.79, H 5.64.
2′-Allyloxy-4,4′-dimethoxychalcone (4). A mixture of chalcone
3 (2.00 g, 7.00 mmol), allyl bromide (1.27 g, 1 mL, 10.50 mmol), and
40% potassium fluoride alumina (5.00 g, 35.00 mmol of KF) in 25 mL
of CH3CN (20 mL) was stirred magnetically at room temperature for
4 h. The solid material was filtered and washed with CH2Cl2. The
filtrate was concentrated under reduced pressure to afford a yellow
residue. Purification of the residue was done by flash chromatography
using petroleum ether−ethyl acetate (5:1) to afford a yellow solid of
chalcone 4 in 85% yield: 1H NMR (400 MHz, CDCl3) δ 3.85 (3H, s,
OCH3), 3.86 (3H, s, OCH3), 4.62 (2H, dd, J = 5.0, 1.0 Hz, CH2−
allyyl), 5.28 (1H, dd, J = 10.3, 1.3 Hz, allyl), 5.47 (1H, dd, J = 17.3,
1.3, allyl), 6.07 (1H, m, allyl), 6.48 (1H, d, J = 1.3 Hz, H-3′), 6.58
(1H, dd, J = 8.0, 1.3 Hz, H-5′), 6.91 (2H, d, J = 8.7 Hz, H-3, H-5),
7.46 (1H, d, J = 16.0 Hz, H-α), 7.78 (1H, d, J = 8.0 Hz, H-6′), 7.68
(1H, d, J = 16.0 Hz, H-β), 7.78 (2H, d, J = 8.7 Hz, H-2, H-6); 13C
NMR (100 MHz, CDCl3) δ 55.3 (OCH3), 55.5 (OCH3), 69.4 (allyl),
99.8 (C-3′), 105.5 (C-5′), 114.2 (C-3, C-5), 117.8 (C-3), 122.8 (C-
α), 125.1 (C-1′), 128.1 (C-2), 129.7 (C-2, C-4), 132.4 (C-1), 133.8
(C-6′), 141.6 (C-β), 159.2 (C-4), 161.1 (C-2′), 164.8 (C-4′), 190.4
(CO); HRESIMS m/z [M + H]+ 325.1415 (calcd for C20H21O4,
325.1434); anal. calcd for C20H20O4, C 74.06, H 6.22; found: C 74.03,
H 6.19.
General Procedure for the Synthesis of Chalcones 6−15.
The chemistry of chalcones 6−15 was already published by our
group.36 A mixture of equimolar amounts of appropriate acetophe-
nones and benzaldehydes was added to a round-bottom flask in 25
mL of EtOH. Then a 15 N ethanolic solution of sodium ethoxide (0.2
mL) was added to the reaction mixture, and the reaction mixture was
stirred overnight (15−17 h) at room temperature. Then the solvent
was removed, and the mixture was dissolved in ethyl acetate. The
contents of the flask were then transferred to the separating funnel
and washed with brine. Then the organic layer was dried over MgSO4,
and the solvent was removed to obtain the product. In case the
product was an oil, it was purified by column chromatography in
EtOAc−hexanes, and in case of the solid, it was purified by
recrystallization from ethanol to afford chalcones.
Combinations of FLT3 inhibitors with chemotherapeutic or
targeted drugs have been used in the clinic for enhanced
efficacy and to overcome resistance to FLT3 inhibitors.22,51
Despite their advantages, fully elucidating clinical benefits with
combination can be challenging, considering the pharmacoki-
netic differences such as drug−drug interactions.52 Use of
multitarget monotherapies (polypharmacology) provides an
alternative to drug combinations,53 and many small molecule
inhibitors with numerous targets in addition to FLT3 have
been reported to overcome TKD-mediated resistance to FLT3
inhibitors.23 Examples include dual inhibitors of FLT3/CDK4
(AMG 925),54 FLT3/JAK2 (pacritinib),55 and FLT3/Aurora
(CCT137690).26 Similarly, some kinase inhibitors have shown
microtubule depolymerizing activity, which could contribute to
their enhanced efficacy and ability to evade acquisition of drug
resistance.56 A microtubule polymerization inhibitor with
activity against multiple kinases, including FLT3, has been
shown to inhibit proliferation and induce apoptosis in cancer
cell lines of various origins including leukemia.34 Chalcone 4
identified in this study represents a promising lead for targeting
a subset of AML as a single agent. Further lead optimization,
selectivity, and pharmacokinetic studies are required for in vivo
evaluation of chalcone 4.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
measured on a Yanaco MP-J3 model melting point apparatus. The
NMR spectra were recorded using an Avance 500 MHz spectrometer
(Bruker, Billerica, MA, USA) or Agilent DD2 400 system (Agilent
Technologies, USA). Chemical shifts are reported in ppm (δ).
Coupling constants, J, are reported in Hz. HRESIMS were obtained
on a Thermo Fischer LTQ Orbitrap Elite mass spectrometer, while
elemental analyses were recorded on an Elementar Vario EL CHNS-
O elemental analyzer. Column chromatography was performed on
Sephadex LH 20 (Fluka, Germany), silica gel (0.040−0.063 mm,
Merck, Germany), and silica gel 60 silanized (0.063−0.200 mm,
Merck, Germany), whereas analytical TLC was performed on
precoated silica gel F 254 aluminum sheets (Merck, Germany).
Yields refer to chromatographically and spectroscopically pure
compounds unless otherwise stated. All reagents and solvents were
purchased from commercial sources unless otherwise specified.
General Procedure for the Synthesis of Chalcones 1−3. A
mixture of equimolar amounts of acetophenones, viz., 1-(2-
hydroxyphenyl)ethanone or 2-hydroxy-4-methoxyacetophenone, and
benzaldehydes, such as benzaldehyde, 3,4-dimethoxybenzaldehyde, or
4-methoxybenzaldehyde, in 25 mL of EtOH was heated at 50 °C.
After the addition of 50% aqueous NaOH (6 mL) solution, the
mixture was further stirred at 50 °C for 5 h. Then, the reaction
mixture was stirred at room temperature for 24 h, and a yellow
precipitate was formed. Furthermore, each mixture was diluted with
ice-cold water (150 mL) and then acidified with diluted HCl (20
mL). Finally, the resulting precipitates formed were filtered off, dried,
and crystallized with aqueous ethanol to afford the desired chalcone.
2′-Hydroxychalcone (1). Chalcone 1 was prepared according to
a general procedure, and spectroscopic data were in good agreement
with previously published data.57 1H NMR (500 MHz, CDCl3) δ 6.95
(1H, m), 7.04 (1H, dd, J = 1.5, 8.0 Hz), 7.04 (1H, dd, J = 1.5, 8.0
Hz), 7.49 (3H, m), 7.51 (1H, m), 7.67 (3H, m), 7.95 (2H, m), 12.81
Cell Culture. MV-4-11, MOLM-13, MOLM-13-Res (provided by
Dr. Spiros Linardopoulos, Institute of Cancer Research, London,
UK), K-562, and THP-1 were cultured in Roswell Park Memorial
Institute (RPMI) medium, while HCT116 and A549 cells were
cultured in DMEM (Dulbecco’s modified Eagle’s medium). Both
media were supplemented with 10% fetal bovine serum (FBS) and 5%
antibiotic−antimycotic. All the cell lines in their respective
supplemented medium were cultured at 37 °C in humidified
incubators with 5% CO2.
Cell Proliferation Assay. Effect of compounds on the
proliferation of cells was measured through a colorimetric MTS cell
proliferation assay. For initial screening, 15 000 cells (MV-4-11 and
K562) were split per well in 96-well plates, treated with 25 and 50 μM
concentrations of the compounds, and incubated at 37 °C for 72 h. At
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the end of treatment, 20 μL of MTS solution was added to each well,
and the plates were again incubated at 37 °C for 3 h. Optical density
(OD) was then measured at 490 nm on a microplate reader.
Percentage inhibition was calculated, and graphs were made in Excel.
For GI50 determinations, 15 000 cells per well in 96-well plates were
treated with nine 2-fold dilutions of the inhibitors (starting with 50
μM) and incubated at 37 °C for 72 h. At the end of the treatment, the
MTS assay was performed as described above, and the GI50 values
were calculated using GraphPad Prism.
Immunoblotting. Cells treated with the indicated concentrations
of chalcone 4 for 8 or 24 h were collected in lysis buffer (25 mM Tris
pH 7.4, 1% Triton X-100, 50 mM sodium chloride supplemented with
protease and phosphatase inhibitors) through incubation on ice for 10
min. Cell lysates were sonicated and cleared by centrifugation at
13600g for 10 min. Protein concentrations of the cleared cell lysates
were measured, and equal amounts of proteins were used for
preparing the samples for SDS-PAGE. Samples were run on 10%
SDS-PAGE, and separated proteins were transferred onto nitro-
cellulose membranes. Membranes were blocked with 5% skimmed
milk and incubated overnight at 4 °C with the primary antibodies.
The next day, membranes were washed with PBS containing 0.1%
Tween (PBST) and incubated with HRP-labeled secondary antibod-
ies for 1 h at room temperature. The membranes were washed again,
developed with the ECL reagent, and imaged using ChemiDoc.
Antibodies for Stat5, P-Stat5, cleaved PARP, and cleaved caspase 3
were from Cell Signaling Technology (CST). Antibodies for P-
histone H3 (S10) and alpha tubulin were from Abcam, P-Erk and
GAPDH were from Santa Cruz, and T-histone H3 were from
Southern Biotech.
Microtubule Repolymerization Assay. For the immunofluor-
escence-based microtubule repolymerization assay, A549 cells were
plated on P-L-lysin-coated coverslips in six-well plates. Cells were
treated (in two sets) with the different concentrations of inhibitors or
DMSO control and incubated on ice for 30 min for microtubule
depolymerization. One set of cells was fixed with ice-cold methanol,
and the other set was then shifted to 37 °C for 10 min to allow
repolymerization of the microtubules followed by fixation with ice-
cold methanol. Fixed cells were blocked with 2% BSA (in PBS) and
incubated with alpha-tubulin antibodies (1:500) for 1 h. Coverslips
were washed with PBS and incubated with FITC-labeled anti-mouse
antibodies (1:500) for 1 h in the dark. Cells were washed five times in
PBS and stained with DAPI (1:10000 dilution) during the
penultimate wash. Stained coverslips were then mounted onto glass
slides with Fluoromount and imaged using a Nikon confocal
microscope.
Cell Cycle and Apoptosis Assay. For the cell cycle analysis, cells
were treated with different concentrations of chalcone 4 for 8 h,
collected through trypsinization, and fixed with 85% ice-cold ethanol.
Fixed cells were washed with PBS containing 1% FBS and stained
with a propidium iodide/RNase solution for 30 min at 37 °C. Cells
were analyzed with a BD FACSCalibur instrument. For the apoptosis
assay, cells were treated with different concentration of chalcone 4 for
24 and 48 h. Cells were then collected and stained with an annexin V/
PI apoptosis kit (Santa Cruz) according to the manufacturer’s
instructions. Stained samples were then analyzed with the BD
FACSCalibur instrument.
temperature. Membranes were developed with ECL reagent on
Chemidoc (Bio-Rad).
In Vitro Assays. Both an in vitro tubulin polymerization assay and
a colchicine displacement assay were performed by Ecrins
Therapeutics (France), as described elsewhere.60 The FLT3 kinase
assay was carried out by SelectScreen Kinase Profiling Services
(ThermoFisher).
Docking Studies. AutoDock Tools (ADT, The Scripps Research
Institute, La Jolla, CA, USA) was used to perform the docking. The
crystal structure of FLT3 was obtained from Protein Data Bank (PDB
code: 4rt7). It was prepared for docking by removing the ligand. The
addition of hydrogen atoms was performed using MGLTools for
AutoDock, and the docking was performed at an exhaustiveness of 8
and grid box dimensions of 40, 40, 40. The docking for tubulin was
performed as described by Manzoor et al.60 Analysis of the docking
was done by checking the interaction energy, clustering, and the
conformation of the protein and ligand using PyMol and Discovery
Studio Visualizer.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00699.
■
sı
Supplementary figures and detailed NMR data (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Amir Faisal − Department of Biology, Syed Babar Ali School of
Science and Engineering, Lahore University of Management
Sciences, Lahore, Pakistan; orcid.org/0000-0002-7376-
8286; Phone: +92-423-560-8453; Email: amir.faisal@
lums.edu.pk
Hidayat Hussain − Department of Chemistry, University of
Paderborn, 33098 Paderborn, Germany; Department of
Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry,
D-06120 Halle (Saale), Germany; orcid.org/0000-0002-
8654-8127; Email: hussainchem3@gmail.com,
Hidayat.Hussain@ipb-halle.de
Authors
Haleema Sadia Malik − Department of Biology, Syed Babar Ali
School of Science and Engineering, Lahore University of
Management Sciences, Lahore, Pakistan
Aishah Bilal − Department of Biology, Syed Babar Ali School of
Science and Engineering, Lahore University of Management
Sciences, Lahore, Pakistan
Rahim Ullah − Department of Biology, Syed Babar Ali School of
Science and Engineering, Lahore University of Management
Sciences, Lahore, Pakistan
Maheen Iqbal − Department of Biology, Syed Babar Ali School
of Science and Engineering, Lahore University of Management
Sciences, Lahore, Pakistan
Sardraz Khan − Department of Chemistry and Chemical
Engineering, Syed Babar Ali School of Science and Engineering,
Lahore University of Management Sciences, Lahore, Pakistan
Ishtiaq Ahmed − Department of Chemistry, University of
Paderborn, 33098 Paderborn, Germany
Karsten Krohn − Department of Chemistry, University of
Paderborn, 33098 Paderborn, Germany
Dot-Blot Screening. For dot-blot screening, 50 000 HCT116
cells per well were split in a 96-well plate on day 1. The next day, cells
were treated with 25 μM concentrations of the test compounds for 8
h. Cells were then lysed by adding 40 μL of 2× SDS sample buffer
containing 200 mM dithiothreitol (DTT). Cells in each well were
sonicated for 5 s followed by incubation of the plate on a hot-plate for
10 min. The following day, 2.5 μL of the samples was blotted on
nitrocellulose membranes and dried for 20 min and then washed once
with PBST for 2 min. The membranes were blocked in 5% milk. Anti-
phospho-histone H3 antibody (1:500 dilution) was added and
incubated overnight at 4 °C. The membranes were given three 5 min
washes with PBST, then incubated with the anti-rabbit secondary
antibody (1:1000 dilution, Southern Biotech) for 1 h at room
Rahman Shah Zaib Saleem − Department of Chemistry and
Chemical Engineering, Syed Babar Ali School of Science and
Engineering, Lahore University of Management Sciences, Lahore,
Pakistan; orcid.org/0000-0002-9615-4471
Complete contact information is available at:
I
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A.; Brandwein, J. M.; Witte, T.; Niederwieser, D.; Appelbaum, F. R.;
Medeiros, B. C.; Tallman, M. S.; Krauter, J.; Schlenk, R. F.; Ganser,
A.; Serve, H.; Ehninger, G.; Amadori, S.; Larson, R. A.; Dohner, H. N.
Engl. J. Med. 2017, 377, 454−464.
https://pubs.acs.org/10.1021/acs.jnatprod.0c00699
Notes
The authors declare no competing financial interest.
(25) Larrosa-Garcia, M.; Baer, M. R. Mol. Cancer Ther. 2017, 16,
991−1001.
ACKNOWLEDGMENTS
■
(26) Moore, A. S.; Faisal, A.; Gonzalez de Castro, D.; Bavetsias, V.;
Sun, C.; Atrash, B.; Valenti, M.; de Haven Brandon, A.; Avery, S.;
Mair, D.; Mirabella, F.; Swansbury, J.; Pearson, A. D.; Workman, P.;
Blagg, J.; Raynaud, F. I.; Eccles, S. A.; Linardopoulos, S. Leukemia
2012, 26, 1462−1470.
This research was supported by the startup grant (STG-BIO-
1009) provided by Lahore University of Management Sciences
(LUMS) to A.F. H.H. thanks the Alexander von Humboldt
Foundation for its generous support in providing the
opportunity to do research in Germany
(27) Bavetsias, V.; Crumpler, S.; Sun, C.; Avery, S.; Atrash, B.; Faisal,
A.; Moore, A. S.; Kosmopoulou, M.; Brown, N.; Sheldrake, P. W.;
Bush, K.; Henley, A.; Box, G.; Valenti, M.; de Haven Brandon, A.;
Raynaud, F. I.; Workman, P.; Eccles, S. A.; Bayliss, R.; Linardopoulos,
S.; Blagg, J. J. Med. Chem. 2012, 55, 8721−8734.
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