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
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