Unnatural polyketide analogues selectively target the her signaling pathway in human breast cancer cells

Article abstract of DOI:10.1002/cbic.200900674

Receptor tyrosine kinases are critical targets for the regulation of cell survival. Cancer patients with abnormal receptor tyrosine kinases (RTK) tend to have more aggressive disease with poor clinical outcomes. As a result, human epidermal growth factor

Full text of DOI:10.1002/cbic.200900674

DOI: 10.1002/cbic.200900674
Unnatural Polyketide Analogues Selectively Target the
HER Signaling Pathway in Human Breast Cancer Cells
Seok Joon Kwon,[a] Moon Il Kim,[a] Bosung Ku,[a] Lydie Coulombel,[a] Jin-Hwan Kim,[b]
Joseph H. Shawky,[a] Robert J. Linhardt,[b] and Jonathan S. Dordick*[a]
Receptor tyrosine kinases are critical targets for the regulation
of cell survival. Cancer patients with abnormal receptor tyro-
sine kinases (RTK) tend to have more aggressive disease with
poor clinical outcomes. As a result, human epidermal growth
factor receptor kinases, such as EGFR (HER1), HER2, and HER3,
represent important therapeutic targets. Several plant polyphe-
nols including the type III polyketide synthase products (genis-
tein, curcumin, resveratrol, and epigallocatechin-3-galate) pos-
sess chemopreventive activity, primarily as a result of RTK in-
hibition. However, only a small fraction of the polyphenolic
structural universe has been evaluated. Along these lines, we
have developed an in vitro route to the synthesis and subse-
quent screening of unnatural polyketide analogues with N-ace-
tylcysteamine (SNAc) starter substrates and malonyl-coenzy-
me A (CoA) and methylmalonyl-CoA as extender substrates.
The resulting polyketide analogues possessed a similar struc-
tural polyketide backbone (aromatic-2-pyrone) with variable
side chains. Screening chalcone synthase (CHS) reaction prod-
ucts against BT-474 cells resulted in identification of several tri-
fluoromethylcinnamoyl-based polyketides that showed strong
suppression of the HER2-associated PI3K/AKT signaling path-
way, yet did not inhibit the growth of nontransformed MCF-
10A breast cells (IC50 >100 mm). Specifically, 4-trifluoromethyl-
cinnamoyl pyrone (compound 2e) was highly potent (IC50 <
200 nm) among the test compounds toward proliferation of
several breast cancer cell lines. This breadth of activity likely
stems from the ability of compound 2e to inhibit the phos-
phorylation of HER1, HER2, and HER3. Therefore, these poly-
ketide analogues might prove to be useful drug candidates for
potential breast cancer therapy.
Introduction
The human epidermal growth factor receptor (HER) family,
which includes EGFR (HER1), and HER2, and HER3 serve as im-
portant targets for selective cancer therapy.[1] Overexpression
of HER2 is found in ~30% of human breast cancers and is as-
sociated with more aggressive and drug resistant tumors.[2] Di-
merization of the HER receptors (HER1/HER2, HER2/HER2, and
HER2/HER3) induces autophosphorylation of specific tyrosine
residues within the cytoplasmic catalytic kinase domain of the
activated receptor.[3] These tyrosine autophosphorylation resi-
dues activate downstream cell proliferation (mitogen-activated
protein kinase, MAPK) and the survival (phosphatidylinnositol-3
kinase, PI3K/Akt[4]) signaling pathway. HER2 is also the pre-
ferred heterodimeric partner with other HER receptors. Thus,
HER2 is an attractive target for cancer drug development.
Several small molecule tyrosine kinase (TK) inhibitors, such
as lapatinib, gefitinib, and erlotinib, which target the intracellu-
lar kinase domain of the HER receptor, are clinically available.[5]
However, these compounds have toxic side effects, including
skin rash, diarrhea, and thrombocytopenia.[6] In addition, in
some lung cancer patients treated with gefitinib and erlotinib,
somatic mutations were observed in the EGFR kinase
domain;[7] this resulted in altered drug sensitivity and increased
drug resistance.[8] Thus, development of safer, yet highly
potent and selective TK inhibitors is needed.
stimulating apoptosis in cancer cells, and appear to serve as
chemopreventive agents against oxidative damage in normal
cells.[9] The diverse biological activity of polyphenolic-based
polyketides relates to the ability of these structurally complex
molecules to activate or inhibit one or more molecular targets
that are critical in cancer cell proliferation and/or metastasis.[9]
In addition, these natural products are generally nontoxic and
have been used clinically for targeting HER2,[10] cyclooxyge-
nase-2,[11] nuclear factor kB (NF-kB) transcription factor,[12] pro-
tein kinases (e.g., Akt and MAPKs),[13] Bcl-2,[14] among others.
Despite the large number of polyphenols that exist in
nature, only a small fraction of the polyketide and polyphenol-
ic structural universe can be tapped because of difficulties in
[a] Dr. S. J. Kwon, Dr. M. I. Kim, Dr. B. Ku, Dr. L. Coulombel, J. H. Shawky,
Prof. J. S. Dordick
Department of Chemical and Biological Engineering,
Center for Biotechnology and Interdisciplinary Studies,
Rensselaer Polytechnic Institute
Troy, NY 12180 (USA)
Fax: (+1)5182762207
E-mail: dordick@rpi.edu
[b] Dr. J.-H. Kim, Prof. R. J. Linhardt
Department of Chemistry and Chemical Biology,
Center for Biotechnology and Interdisciplinary Studies,
Rensselaer Polytechnic Institute
Products of the type III polyketide synthases, including poly-
phenols such as catechins, genistein, resveratrol, and curcumin,
possess anticancer and antiangiogenic activity, are capable of
Troy, NY 12180 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.200900433
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J. S. Dordick et al.
isolation of these compounds. Diversity-oriented synthesis
(DOS) of natural product-like polyphenolic compounds[15] and
de novo polyphenol synthesis by plant-based type III poly-
ketide synthases (PKS) are possible alternative routes to the
generation of novel polyphenols. Although DOS has great po-
tential to generate diverse natural product-like architectures,
multiple steps are required for polyketide chain elongation
and cyclization,[16] whereas, type III PKS alone is capable of per-
forming both steps. Exploiting these PKSs to synthesize natural
and unnatural polyphenols is possible and might be expected
to yield unexplored structurally diverse natural products.[17]
Many secondary metabolism pathways serve as excellent can-
didates for this strategy, including polyketides, isoprenoids,
lignans, etc., and rapid microarray-based screens can be devel-
oped based on key targets, e.g., caspase family enzymes, kin-
ases, phosphatases, histone deacetylases, as well as cell-based
assays.[18] The polyketide synthases are particularly well-suited
for in vitro manipulation. The type III PKS are fundamentally
the simplest among the three classes of PKS, and are com-
prised of homodimeric enzymes that catalyze decarboxylative
condensation of malonyl-CoA or methylmalonyl-CoA onto a
wide range of starter-CoA substrates. In nature, these enzymes
catalyze the synthesis of a diverse array of flavonoids, chal-
cones, stilbenes, and together with other enzymes, a large
number of hybrid molecules with activities as varied as anti-in-
fectives to anticancer agents.[19] Moreover, examples of natural
post-PKS tailoring reactions are known,[20] and our previous ef-
forts have led to hybrid natural–unnatural polyketide synthesis
through post-PKS tailoring reactions.[18a]
Results and Discussion
In Vitro Synthesis of Unnatural Polyketides
To obtain novel (unnatural) polyphenols derived from Type III
PKS, we performed in vitro precursor-directed synthesis with
CHS from alfalfa (Medico sativa) and various N-acetylcystea-
mine (SNAc) thioester starter substrates (Figure 1S), with ma-
lonyl-CoA or methylmalonyl-CoA serving as extender sub-
strates (Scheme 1). To ensure that CHS reacted with SNAc
esters in a manner similar to the respective CoA esters, a stan-
dard reaction was performed by using p-coumaryl-SNAc and
malonyl-CoA as starter and extender substrates, respectively.
HPLC-MS/MS measurement of the reaction mixture resulted in
two peaks with parent ions [M+H]+ at m/z 273 and [MÀH]À at
m/z 271, indicating the successive condensation of three units
of malonyl-CoA. Based on MS/MS-ESI fragmentation analysis,
one fragment at m/z 227 corresponded to [MÀHÀCO2]À, and
this indicates the presence of an a-pyrone ring (3b), which
was terminated without aromatic ring formation. The second
peak gave a similar UV spectrum (lmax 296 nm) and mass spec-
trum identical to the expected product naringenin (4b). Thus,
the SNAc starter substrate was effectively used by CHS to gen-
erate the natural product of the reaction.
Encouraged by this result, we proceeded to perform CHS
catalysis on a range of starter substrates with both malonyl-
CoA and methylmalonyl-CoA as extender substrates
(Scheme 1). The products from 21 enzymatic reactions were
analyzed by HPLC-MS, and this resulted in the identification of
a large number of structurally diverse products, including tri-
and tetraketide pyrones and fully formed flavonoids. 1H and
13C NMR analyses were performed on seven of these products,
thus confirming the MS identification.
Chalcone synthase (CHS) is the key enzyme involved in poly-
phenol biosynthesis and possesses a very wide substrate spe-
cificity,[21] and utilizes both aromatic and aliphatic CoA esters
as substrates.[22] The natural function of CHS is to initiate poly-
phenol biosynthesis through sequential Claisen condensation
of coumaroyl-CoA as a starter substrate with malonyl-CoA as a
chain extender substrate. After three rounds of chain elonga-
tion, the resulting linear tetraketide intermediate is cyclized to
form the chalcone naringenin. In some cases, immature poly-
phenols are produced through truncated condensation reac-
tions to yield triketide and tetraketide products that are then
cyclized to produce coumaroyl-4-hydroxy-2-pyrones.[20] CHS
possesses a high degree of substrate promiscuity, including
both aromatic and aliphatic CoA esters as substrates,[22] albeit
far less promiscuity is evident for the extender substrate.[21,23]
We reasoned that such broad specificity could be used to facil-
itate the discovery of new molecular entities with potent bio-
logical activities.
Inhibitory Effect of CHS Reaction Products on the HER2-
PI3K-Akt Signaling Pathway
CHS reaction products were then extracted from the reaction
mixtures into ethyl acetate and evaluated as inhibitors of the
HER2-associated PI3K/Akt signaling pathway. HER2-overex-
pressing BT-474 cells[24] were then used to investigate whether
the polyketide products suppressed HER2 signaling. The ex-
tracted reaction mixtures were redissolved in DMSO. The re-
sulting product mixtures were used in 2D BT-474 cell culture
assays in 6-well plates. The inhibitory activity of these product
mixtures were assessed against the HER2-PI3K-Akt signaling
pathway (Figure 1 and 2). As a negative control, the vehicle
containing 1% (v/v) DMSO was used and it showed the ex-
pected high levels of phospho-Akt (p-Akt), which is the down-
stream output of HER2 activation; thus, this demonstrated the
active PI3K-Akt signaling cascade. Positive controls consisted
of 2 mm wortmannin and 50 mm LY-294002, which are known ir-
reversible PI3K inhibitors.[25] The reaction mixtures derived
from 2-, 3-, and 4-trifluoromethylcinnamoyl-SNAc starter sub-
strates (1c–1 f) appeared to strongly inhibit formation of phos-
pho-HER2 (p-HER2), which shuts down the downstream kinase
signaling pathway such that negligible phosphorylation of
In the current work, we used an in vitro precursor-directed
synthesis strategy employing CHS with a range of structurally
diverse aromatic starter substrates to generate compounds
that possessed nanomolar activity against proliferation of sev-
eral HER2-overexpressing breast cancer cell lines. Importantly,
these compounds were inactive against a nontumorigenic
breast cell line (MCF-10A), thereby providing highly selective
and potent biological activity with potential therapeutic appli-
cations.
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Unnatural Polyketide Analogues as HER Tyrosine Kinase Inhibitors
Scheme 1. CHS-catalyzed synthesis of unnatural polyphenols from cinnamoyl-SNAc starter substrates with a) malonyl-CoA and b) methylmalonyl-CoA as ex-
tender units. Isolated yields of compounds 2–6 ranged from 5–17%.
PI3K and Akt occur (Figure 3). This inhibition did not take place
some activity was observed with 50 mm of the 3,5-bis(trifluoro-
methyl)cinnamoyl-SNAc (compound 1 f) and 2-trifluoromethyl-
with 50 mm of the free cinnamic acid derivatives, although
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J. S. Dordick et al.
Figure 3. Effect of trifluoromethylcinnamic acid-based compounds (corre-
sponding acids, SNAc esters, and their CHS reaction extracts) on HER2-medi-
ated PI3K-Akt signaling pathway. The BT-474 cells, which are confluent in 6-
well plates (3 mL), were treated for 3 h with different trifluoromethylcinnam-
ic acids (50 mm), trifluoromethylcinnamoyl-SNAc esters (50 mm), and their
CHS reaction extracts (10 mL). Whole-cell lysates were subjected to SDS-PAGE
analysis and probed with anti-phospho-HER2 (Tyr1221/1222), anti-HER2,
anti-phospho-Akt (Ser473), anti-Akt antibody, anti-phospho-PI3K (Tyr508),
and anti-actin, respectively.
Figure 1. Schematic representation of HER2-mediated PI3K-Akt signaling
pathway. Activation of HER2 initiates the activation of the cytoplasmic tyro-
sine kinase domain and subsequent events to activate downstream of PI3K
and Akt. The activation of Akt suppresses apoptosis and stimulates the cell
survival pathway.
High-Yield Production of Bioactive Polyketide Analogues
using Immobilized CHS
The reaction mixtures from the trifluoromethylcinnamoyl-SNAc
starter substrates (with either malonyl-CoA or methylmalonyl-
CoA as extender substrates) possessed the highest biological
activity. However, these mixtures were highly impure and the
product concentrations were low (<3–5% conversion), as a
result of the instability of CHS under the in vitro reaction con-
ditions (half-life of 5 h). Following our previous work,[26] we im-
mobilized CHS onto Ni-NTA agarose beads; this also served as
a method to purify the enzyme through its N-terminal His8-
tagged construction. This approach resulted in improved CHS
stabilization (half-life of ca. 7 days, data not shown) and ~20%
conversion (Figure 2S in the Supporting Information) for both
2- and 4-trifluoromethylcinnamoyl-SNAc starter substrates, by
using malonyl-CoA as extender, which enabled us to isolate
the triketide pyrone products (compounds 2c and 2e, respec-
tively, structures confirmed by NMR, see Supporting Informa-
tion) after scale-up (50 mL reaction volume).
Figure 2. Effect of precursor-directed CHS reaction extracts on the level of
phospho-Akt in HER2-overexpressing human breast cancer BT-474 cells in 6-
well plate 2D cultures. Abbreviation: C, control (without treatment); LY,
LY294001; W, wortmannin; M, malonyl-CoA; MM, methylmalonyl-CoA.
Whole-cell lysates were subjected to SDS-PAGE analysis and probed with
anti-phospho-Akt (Ser473) and anti-Akt antibody, respectively.
Cell Specific Growth Inhibition Assays by Bioactive Poly-
ketide Analogues
Shutting down the HER2 signaling pathway was expected to
activate apoptosis, which would lead to growth inhibition[1] at
increased doses of the CHS products. This was tested by using
purified compounds 2c and 2e for dose response assays
against BT-474 cells. As shown in Figure 3S (Supporting Infor-
mation), both compounds showed strong growth inhibition
activity against BT-474 cells, with IC50 values of 120 and 30 nm,
respectively (Table 1). Interestingly, neither compound was
active against nontransformed MCF10 A breast cells (IC50 >
100 mm, Table 1).
cinnamoyl-SNAc (compound 1c) starter substrates (reduced
levels of p-Akt generated). Therefore, only in the presence of
the full CHS product mixtures was there significant inhibitory
activity. These results were not influenced by the effects of cell
concentration, as the actin signal remained relatively constant
(Figure 3).
The trifluoromethyl group is critical for inhibitory activity.
When the CF3 group of compound 2e is replaced with the CH3
group of compound 2g, an 1800-fold loss in inhibitory
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Unnatural Polyketide Analogues as HER Tyrosine Kinase Inhibitors
alogue (compound 2g) and much lower apoptosis and PARP
cleavage was evident with the SNAc starter substrate (com-
pound 1e); this is consistent with the trifluoromethyl-contain-
ing pyrone as being a highly active HER2-pathway inhibitor.
Due to the highly potent growth inhibition activity of com-
pound 2e towards BT-474 cells, we performed similar experi-
ments for both compound 2e and a known HER2 specific in-
hibitor, AG-825,[27] against two other HER2-overexpressing
human breast cancer cell lines, including SKBR-3[28a] and trastu-
zumab (Herceptin)-resistant MDA-MB-453 cells[28] (Figure 5a).
Table 1. Cell growth inhibition by 2- and 4-trifluoromethylcinnamoyl-
SNAc (1c and 1e), their corresponding pyrones (2c and 2e), and 4-meth-
ylcinnamoyl pyrone (2g) on human normal (MCF-10 A) and tumor-
derived cell lines (MCF-7 and BT-474).
IC50 [mm]
Cell line
BT-474
MCF-7
MCF-10A
Compounds
Compound 2e
Compound 1e
Compound 2c
Compound 1c
Compound 2g
0.03Æ0.001
1.0Æ0.1
0.3Æ0.02
>100
>100
>100
>100
>100
1.5Æ0.2
3.1Æ0.1
15Æ0.2
0.12Æ0.02
5.5Æ0.2
55Æ1.5
>100
activity is obtained (Table 1). The pyrone component is also im-
portant for inhibitory activity; the SNAc starter substrates were
less inhibitory than the pyrone products (ca. 30-fold lower ac-
tivity for compound 1e vs. compound 2e).
In addition to dramatic growth inhibition, compound 2e
strongly activated apoptosis in BT-474 cells, as demonstrated
by severe cellular morphological changes and disruption of the
cell membrane. This was reflected in increasing red-colored
cells (dead cells) by labeling nuclei with membrane-impermea-
ble ethidium homodimer-1 (Figure 4a, insets). Poly(ADP-ribose)
polymerase (PARP) cleavage to an 89 KDa fragment, the hall-
mark of apoptosis, was also shown to be strongly activated by
compound 2e (Figure 4b). No evidence of apoptosis (as indi-
cated by lack of PARP cleavage) was obtained with the CH3 an-
Figure 5. A) Dose-dependent effect of 4-trifluoromethylcinnamoyl pyrone
(2e) and AG-825 on growth inhibition of different breast cancer cell lines
(MCF-7, BT-474, SK-BR-3, MDA-MB-453) and a nontransformed breast cell
line, MCF-10A. B) Dose response curves of HER1 and HER2 kinase activity by
4-trifluoromethylcinnamoyl pyrone (2e) and AG-825. Enzyme inhibition was
assessed by measuring the inhibition of biotinylated peptide substrate phos-
phorylation (DNEY*FY*V and DNDY*INA were used as substrates for HER2
and HER1, respectively)
Compound 2e was highly potent against both cell lines with
IC50 values of 60 and 130 nm against SKBR-3 and MDA-MB-453,
respectively, whereas AG-825 was less potent against these cell
lines (IC50 values of BT-474, SKBR-3 and MDA-MB-453 were
0.84, 2.7 and 132 mm, respectively). Trastuzumab-resistant
MDA-MB-474 cells showed the lowest sensitivity against AG-
825. When we compared the growth inhibition of compound
2e with commercially available multiple-kinase inhibitor drug,
lapatinib (Tykerb), lapatinib had IC50 values against SKBR-3 cells
of 37–76 nm[29] and IC50 values of 2.5–71 nm against BT-474
cells.[29] Thus, the effect of growth inhibition of compound 2e
was similar to commercially available lapatinib.
Figure 4. Effect of 4-trifluoromethyl cinnamoyl SNAc (1e) and 4-trifluoro-
methyl- cinnamoyl pyrone (2e) on apoptosis of BT-474 cells. Cells were
treated with compounds 1e or 2e (each 5 mm) for 12 h with PDGF. After
treatment, apoptosis was detected by: A) the live (green)/dead (red) assay
(inset) and cell morphological changes, and B) p-Her2 (Tyr1221/1222), p-Akt
(Ser473), and cleaved PARP signals
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To gain a preliminary mechanistic understanding of the
effect of compound 2e, we evaluated the ability of the com-
pound to inhibit HER1 and HER2 TK by using an ELISA assay
(Figure 5b). Compound 2e was a potent inhibitor of both
HER1 and HER2 TK, with IC50 values of 0.33 and 0.16 mm, re-
spectively. This contrasts with AG-825, which had IC50 values of
23 and 0.59 mm for HER1 and HER2, respectively, thus indicat-
ing potent HER2 and only moderate HER1 TK inhibition. In ad-
dition, phosphorylation of kinase domain residue Tyr877 of
HER2 was also inhibited by compound 2e, as well as AG-825
(Figure 4 in the Supporting Information). These results demon-
strate that compound 2e is an effective dual inhibitor of the
TK function of HER1 and HER2. This, in turn, enables growth in-
hibition against recalcitrant cell lines. It is important to note
that while compound 2e is effective against MDA-MB-453
cells, this cell line is resistant to Herceptin (trastuzumab).
CH3 resulted in a compound with an IC50 value of 55 mm—over
1800-fold less active than the CF3-containing analogue. The
high activity of compound 2e was presumably due to potent
inhibition of the HER2 receptor TK, and this was demonstrated
by testing the compound against isolated HER2 kinase. The re-
sulting IC50 value of 160 nm was substantially lower than that
of compound 2g (9.4 mm, data not shown); this indicates that
the CF3 group is critical for inhibition of TK activity. In addition
to the CF3 group, the pyrone moiety is important. Comparison
of compound 2e with its relevant SNAc starter substrate (com-
pound 1e) revealed that the latter is approximately 30-fold
less active than the former. As a result of the HER2 TK inhibi-
tion, downstream signaling is diminished. This was evident in
diminished levels of p-HER2, p-Akt, and p-PI3K in the BT-474
cell line. Thus, deactivation of downstream signaling pathways
occurs, presumably involving proapoptotic factors including
p53, BAD, procaspase-9, and Forkhead (FKHR) transcription
factor, as well as activation of transcription factors that involve
cell survival, such as NF-kB, cyclic-AMP response element-bind-
ing protein, etc.[30]
Because MDA-MB-453 cells were resistant to a HER2-specific
inhibitor (AG-825, IC50 =132 mm), these cells were chosen to ex-
amine the comparative effects of compound 2e and AG-825
on the phosphorylation of all three HER isoforms. Treatment of
MDA-MB-453 cells with compound 2e strongly suppressed the
phosphorylation of HER1, HER2, and HER3 (Figure 6). AG-825,
however, did not suppress HER1 and HER3 phosphorylation
(Figure 6); this is consistent with the aforementioned specifici-
ty of this compound against only the HER2 TK. The breadth of
activity of compound 2e against all three HER isoforms might
explain the activity against the Herceptin-resistant MDA-MB-
453 cells. It is possible that compound 2e, by virtue of its
broad specificity, can prevent both homodimerization (HER2/
HER2) and heterodimerization (HER1/HER2 and HER2/HER3), re-
sulting in growth inhibition of trastuzumab-resistant MDA-MB-
453 cells although more experimental proof is needed.
Pyrones are commonly observed in polyketides generated
through biosynthetic pathways of the type II[31] and type III
PKSs,[19,32] and have been found to have activity as HIV inhibi-
tors,[33] acetylcholine esterase inhibitors (treatment of Alzheim-
er’s disease),[34] cholesterol esterase inhibitors (treatment of hy-
percholesterolemia),[35] and protein TK inhibitors (treatment of
cancer).[36] Indeed, it is known that chalcone- or isoflavonoid-
based pyrones appear to strongly interact with the adenine
pocket of the ATP binding site of c-Src TK domains, presuma-
bly through both hydrophobic interactions and hydrogen
bonding with hydrophobic residues (Leu275, Ala295, Leu395)
and (Met343, Glu341, Thr340), respectively, resulting in TK
competitive inhibition.[36] Incorporation of fluorobenzyl- or tri-
fluoromethylbenzyl groups into existing TK inhibitors has been
used to increase the selectivity of TK inhibition in addition to
enhancing the potency of cellular activity, perhaps due to the
enhanced hydrophobic interaction with the hydrophobic resi-
dues in the ATP-binding pocket of TK.[37] For example, the fluo-
robenzyl group of EGFR- and HER2-kinase inhibitors occupies a
cleft formed by the hydrophobic residues (Met766, Leu777,
Thr790, Thr854, and Phe856) in HER1 kinase domain.[37b] The
fluorine moiety might also undergo hydrogen bonding to the
Arg776 and Thr790 in the hydrophobic cleft, and is thus impor-
tant for optimal inhibition of HER1; however, we cannot ex-
clude the possibility that the CF3 moiety activates the pyrone
ring as an electrophile. Further studies are underway wherein
an X-ray structure of the HER kinase domain with compound
2e will be obtained, such that specific interactions of the com-
pound with the ATP binding site can be elucidated.
Structural Features of Pyrones that Inhibit HER Tyrosine
Kinase
The CF3 group (compound 2e) appears to be essential for high
cell-based activity and selectivity, as replacing this group with
Conclusions
In conclusion, we used an in vitro strategy to synthesize a li-
brary of polyketide analogues with alfalfa CHS[38] that em-
ployed twelve different aromatic-SNAc starter substrates with
malonyl- or methylmalonyl-CoA as extender substrates. Testing
of this library resulted in the identification of several unnatural
Figure 6. Inhibition of HER1, HER2, and HER3 in MDA-MB-453 cell lines by
compound 2e and AG-825. Whole-cell lysates were subjected to SDS-PAGE
analysis and probed with anti-phospho-HER1 (Tyr1148), anti-HER1, anti-phos-
pho-HER2 (Tyr1221/1222), anti-HER2, anti-phospho-HER3 (Tyr1197), anti-
HER3, and anti-actin, respectively.
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Unnatural Polyketide Analogues as HER Tyrosine Kinase Inhibitors
hEGF (20 ngmLÀ1; Trevigen, Gaithersburg, MD, USA), cholera toxin
(100 ngmLÀ1; Sigma, C8052), hydrocortisone (500 ngmLÀ1, Sigma),
Pen/Strep (1ꢁ; Cellgro, Herndon, VA, USA), and horse serum (5%;
ATCC, Rockville, MD, USA).
polyketide-based polyphenols that suppressed the HER-PI3K-
Akt signaling pathway, and had multiple HER TK inhibitory ac-
tivity. The potent compounds were highly selective against
breast-cancer cell lines, yet were essentially inactive against a
nontransformed breast-cell lines. Precursor-directed in vivo
combinatorial biosynthesis is well known, yielding hybrid or
“unnatural” natural products.[39] However, cell-based combina-
torial synthesis platforms are subject to variabilities in the per-
meability of precursor substrates into the cells and potential
toxicity of the precursors or their biosynthetic products.[40]
Moreover, for complex products or product mixtures, purifica-
tion is often process limiting. Thus, in vitro enzymatic synthesis
might provide an efficient alternative to cell-based approaches,
particularly in cases in which downstream tailoring reactions
can be used to diversify further the product spectrum.
Immunoblot Analysis: After exposing all of compounds with
PDGF to breast cancer cells for 3 h, proteins from the cell lysates
were separated by SDS-PAGE and transferred to a PVDF mem-
brane. The membrane was blocked with skim milk (5%) in PBS-
Tween 20 (0.1%, v/v) at 48C overnight. The membrane was incu-
bated with a primary antibody (1:500–1:1000; anti-phospho-Akt
(Ser473), anti-Akt, anti-HER1, anti-phospho-HER1 (Tyr1148), anti-
phospho-HER2 (Tyr1221/1222), anti-phospho-HER2 (Tyr877), anti-
HER2, anti-phospho-HER3 (Tyr1197), anti-HER3, anti-phospho-PI3K
(Tyr508), anti-PI3K, anti-PARP, and anti-actin) at 48C overnight. All
antibodies were purchased from Cell Signaling Technology, Beverly,
MA, USA. Horseradish peroxidase conjugated anti-rabbit or anti-
mouse IgG was used as secondary antibody. Immunoreactive pro-
teins were visualized by the chemiluminescence protocol (Super-
Signal West, Pierce, Rockford, IL, USA).
Experimental Section
Chemical Synthesis of N-acetylcysteamine Thioester (SNAc) Ana-
logues: The two-step synthesis of the SNAc analogues (1a–1l,
Figure 1 in the Supporting Information) was carried out according
to a modification of a previously reported method.[41] Briefly, N,N’-
dicyclohexylcarbodiimide (1.2 equiv) was added to starting carbox-
ylic acid (1 equiv) in ethyl acetate (25 mL) and the reaction mixture
was stirred for 1 h followed by the addition of N-hydroxysuccini-
mide (1.2 equiv) at RT. After 2 h, the substituted urea was filtered
off and SNAc (1.2 equiv) was added to the reaction mixture and
stirred for 4 h. The SNAc analogues were purified by using flash
silica gel chromatography and identified by LC-ESI/MS and NMR.
Compound characterization is in Supporting Information.
Growth Inhibition Assays: Cells were grown in 96-well plates (1ꢁ
104 cells/well) overnight, then treated with various concentrations
of purified CHS products, and incubated for an additional 72 h. The
effect of compounds on cell growth was examined by the MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay.
Briefly, MTT solution (20 mL, 5 mgmLÀ1, Sigma) was added to each
well and incubated for 4 h at 378C. The supernatant was aspirated,
and the MTT-formazan crystals formed by metabolically viable cells
were dissolved in DMSO (200 mL). Finally, the absorbance was
monitored by using a multi-well spectrophotometer (SpectraMax,
Molecular Devices, Sunnyvale, CA, USA) at 595 nm.
HER Kinase Assay: Purified HER1 and HER2 TK and the corre-
sponding biotinylated peptide substrates were purchased from
Cell Signaling Technology and the kinase reaction was performed
in duplicate. Various concentrations of trifluoromethylcinnamoyl
pyrones (AG-825 and Compound 2e) were added to the HER
kinase reaction mixture in 96-well polystyrene round-bottomed
plates. The reaction mixture contained MgCl2 (5 mm), MnCl2
(5 mm), Na3VO4 (3 mm), DTT (1.25 mm), ATP (20 mm), peptide sub-
strate (1.5 mm), and HER2 kinase (50 ng) in of HEPES buffer (50 mL,
60 mm, pH 7.5). The reaction was initiated by adding the peptide
substrate and terminated after 30 min incubation at 238C by
adding EDTA (50 mL, 50 mm, pH 8.0). Each reaction mixture (25 mL)
was diluted with deionized water (75 mL), and transferred into a
96-well streptavidin coated plate and incubated at 238C for
60 min. After the plate was washed three times with PBS contain-
ing Tween 20 (0.1%; PBST), primary antibody (100 mL; phospho-Ty-
rosine mAb, p-Tyr-100, 1:1000 in PBST with 1% BSA) was added
and incubated at 238C for 60 min. After the plate was washed
three times with PBST, of horseradish peroxidase-conjugated goat
anti-mouse IgG (100 mL, 1:500 in PBST with 1% BSA) was added
and the plate was reincubated at 238C for 30 min. After the plate
was washed five times with PBST, the TMB (3,3’,5,5“-tetramethyl-
benzidine) substrate was added and the plate was incubated at RT
until color formed. The reaction was terminated by adding H2SO4
(50 mL, 1n) and the plate was read at 450 nm.
Expression and Purification of Chalcone Synthase (CHS): Chal-
cone synthase (CHS) from alfalfa was produced by overexpression
of the corresponding gene by using the pET-28 expression vector
in E. coli. The protein was purified by using Ni-NTA column chro-
matography. Nickel-chelating ligand nitrilotriacetic acid (Ni-NTA)
agarose beads were obtained from Invitrogen (Carlsbad, CA, USA)
to give ~4 mg CHS/g bead.
In vitro Precursor-Directed CHS Enzyme Reaction: Tris-HCl buffer
(100 mL, 50 mm, pH 7.5) was added to the reaction mixtures con-
taining starter substrates (1 mm, 1a–1l), extender unit (3 mm, ma-
lonyl-CoA or methylmalonyl-CoA), and purified CHS (20 mL, ca.
2.0 mgmLÀ1) or Ni-NTA immobilized CHS (40 mg protein). The reac-
tion mixtures were incubated at 378C for 2 h. The reactions were
stopped by adding HCl (5 mL, 5% (v/v) 4n) and the products ex-
tracted into ethyl acetate (100 mL) and analyzed by LC-ESI/MS. To
isolate polyketide products, we performed reactions (50 mL size).
The resulting extracts were dried under nitrogen, redissolved in di-
chloromethane (DCM; 2 mL) containing traces of methanol, and
the products were purified by using flash column chromatography
with DCM/MeOH (from 20:1–3:1 (v/v)) as an eluent. Purified sam-
ples were dissolved in CD3OD (0.5 mL, Sigma, St. Louis, MO, USA)
for NMR analysis (Bruker DPX500, GmbH, Germany). Compound
characterization (2a–6k) is in Supporting Information.
Cells and Cell Culture: The human breast cancer cell lines used in
this study were MCF-7, BT-474, SKBR-3, and MDA-MB-453. All cells
were grown in DMEM (Invitrogen) supplemented with fetal bovine
serum (10%; Hyclone, Waltham, MA, USA) and penicillin/streptomy-
cin (1ꢁPen/Strep). We also used a nontransformed MCF-10A cell
line, which is phenotypically normal and nontumorigenic. MCF-10A
cells were cultured in DMEM, insulin (10 mgmLÀ1; Sigma, I1882),
Live/Dead Cytotoxicity Assay: BT-474 cells were plated in slide
chambers. After treatment with compounds 1e and 2e (5 mm,) for
12 h, ethidium homodimer-1 (10 mL, 2 mm) and calcein AM (5 mL,
4 mm) were added to PBS (2 mL), and 500 mL of this mixture was
applied to each slide containing the cell monolayer. After
incubation for 30 min at RT, the cells stained and visualized with
ChemBioChem 2010, 11, 573 – 580
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
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Acknowledgements
This work was supported by the National Institutes of Health
(ES012619 and GM66712). M.I.K. acknowledges support from a
Korea Research Foundation Grant funded by the Korean govern-
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Keywords: chalcone synthase · inhibitors · N-acetylcysteamine
(SNAc) precursors · polyketides · tyrosine kinases
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Received: November 7, 2009
Published online on January 7, 2010
580
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