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Cells, cell culture, and transfections: NCI-H460, DLD1, and HeLa cell
lines were purchased from the American Type Culture Collection
(Rockville, MD). NCI-H460/R cells were selected originally from NCI-
H460 cells and cultured in a medium containing DOX.[29] DLD1-TxR
cells were selected by continuous exposure to stepwise increasing
concentrations of paclitaxel from DLD1 cells.[30] HeLa cells stably
expressing EGFP-Abl (HeLa EGFP-Abl) were obtained by transfec-
tion of HeLa cells with the pCEFL EGFP ABL wild-type expression
vector[31] using lipofectamine LTX (Life Technologies, Carlsbad, CA),
according to the manufacturer’s instructions, followed by selection
with 2 mgmLꢁ1 G418 (Sigma–Aldrich) for 3 weeks. MDR cancer cell
lines (NCI-H460/R and DLD1-TxR) and their sensitive counterparts
were maintained in RPMI 1640 medium supplemented with 10%
FBS, 2 mm l-glutamine, and 10000 UmLꢁ1 penicillin, 10 mgmLꢁ1
streptomycin, 25 mgmLꢁ1 amphotericin B solution. HeLa EGFP-Abl
were maintained in DMEM supplemented with 10% FBS, 2 mm l-
glutamine, 10000 UmLꢁ1 penicillin, and 10 mgmLꢁ1 streptomycin
and were constantly kept under selective pressure with 2 mgmLꢁ1
G418. Selective medium was replaced with regular growth
medium on the day before experiments. All cell lines were subcul-
tured at 72 h intervals using 0.25% trypsin/EDTA and seeded into
fresh medium at the following densities: 8000 cellscmꢁ2 for NCI-
H460 and DLD1, and 16000 cellscmꢁ2 for NCI-H460/R, DLD1-TxR,
and HeLa EGFP-Abl cells. The pCEFL EGFP-Abl expression vector
was a kind gift from Ricardo Sꢂnchez-Prieto (Universidad de Castil-
la-La Mancha — UCLM).
Experimental Section
Virtual library design and screening
400 polar heads and eight lipophilic tails were downloaded from
the Sigma–Aldrich and Alfa Aesar online catalogues and reacted in
silico to generate the focused virtual library. In silico reactions were
performed using a custom script in Python language based on the
OpenEye OEChem Toolkit libraries.[20] The closed form of BV02 was
also included in the library. The virtual library was then prepared
for docking by means of the LigPrep application of the Schrçding-
er’s Maestro Suite for molecular modeling, maintaining all parame-
ters at their default value.[28]
Molecules were then processed using the docking protocol devel-
oped and refined in our previous studies,[12a,16,17] which led to the
identification of the two effective 14-3-3 PPI inhibitors BV01 and
BV02. After docking and rescoring, the ten most promising mole-
cules were selected for synthesis, based on a combination of
chemical redundancy, visual inspection, and docking score.
Biology
U2foxRELOC translocation assay: The U2foxRELOC assay has been
described previously.[18b,22] Briefly, cells were seeded at a density of
1.0ꢁ105 cellsmLꢁ1 into black-wall clear-bottom 96-well microplates
(BD Biosciences). After 12 h of incubation at 378C with 5% CO2,
2 mL of each sample were transferred from the mother plates to
the assay plates. Cells were incubated in the presence of the com-
pounds for 1 h. The cells were then fixed, and the nuclei were
stained with DAPI (Invitrogen). Finally the plates were washed
twice with 1ꢁ phosphate-buffered saline (PBS) and stored at 48C
before analysis.
Immunofluorescence, confocal microscopy, and intensitometric analy-
sis of fluorescence: Cells were washed with PBS, then fixed with 4%
paraformaldehyde in PBS for 20 min and permeabilized with 0.2%
Triton X-100 in PBS for 15 min. Nuclei were stained with a solution
of 1.5 mm of 4’,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich) in
PBS for 5 min. Coverslips were mounted in fluorescence mounting
medium (Dako). Samples were visualized on a TSC SP5 confocal
microscope (Leica, 5100000750), installed on an inverted LEICA
DMI 6000CS (10741320) microscope and equipped with an oil im-
mersion PlanApo 40ꢁ1.25 NA objective. Images were acquired
using LAS AF acquisition software (Leica). Intensitometric analysis
of fluorescence was performed using the Quantitation Module of
the Volocity software (PerkinElmer Life Science). Briefly, total nucle-
ar EGFP fluorescence, defined as EGFP signal co-staining with DAPI
nuclear dye, was measured in five representative confocal fields for
each experimental condition. The resulting mean values ꢀSD are
expressed as a percentage of nuclear EGFP fluorescence. Five rep-
resentative fields were acquired and analyzed for each sample. Sig-
nificance (p value) was assessed by one-way ANOVA test. Asterisks
were attributed for the following significance values: *p<0.05,
**p<0.01.
Image acquisition and processing: The BD Pathway 855 High Con-
tent Bioimager (BD Biosciences, San Jose, CA) was used for auto-
mated image acquisition. Acquired images were processed using
AttoVision software (BD Biosciences). The Bioimager was equipped
with a 488/10 nm enhanced GFP (EGFP) excitation filter, a 380/
10 nm DAPI excitation filter, a 515 nm long-pass (LP) EGFP emission
filter, and a 435 nm LP DAPI emission filter. Images were acquired
in the DAPI and GFP channels of each well using a 10ꢁ dry objec-
tive. The plates were exposed for 0.066 ms (gain 0) to acquire DAPI
images and 0.85 ms (gain 30) for GFP images. Cells were stained
with DAPI to facilitate autofocusing of the microscope and to aid
in image segmentation. An image algorithm was applied to allow
cell nucleus segmentation based on a local threshold. Our segmen-
tation strategy assumes that the cell cytoplasm surrounds the nu-
cleus. Consequently, cytoplasmic fluorescence intensity was calcu-
lated from all the pixels within a circumferential ring surrounding
the nuclear ring mask. The width of the ring was defined to be
small enough to avoid ambiguities due to irregular cell shape.
Based on the definition of cell compartments, the nuclear and cy-
toplasmic levels of GFP fluorescence were quantified.
SRB assays: Cells grown in 25 cm2 tissue flasks were trypsinized,
seeded into flat-bottomed 96-well tissue culture plates (2000 cells
per well for NCI-H460 and DLD1 cells, 4000 cells per well for NCI-
H460/R and DLD1-TxR cells) and incubated overnight. Treatment
with DOX (2.5–50 nm for NCI-H460 and 100–2500 nm for NCI-
H460/R), GSK690693 (5–100 mm), and 9 and 10 (5–100 mm) lasted
72 h. The combined effects of 9 and 10 (10 mm) with DOX or
GSK690693 were studied in simultaneous treatments. The cell
growth inhibitory assay was performed after 72 h. The cellular pro-
teins were stained with SRB, following slightly the modified proto-
col of Skehan et al.[32] Briefly, the cells in 96-well plates were fixed
in 50% trichloroacetic acid (50 mL per well) for 1 h at 48C, rinsed in
tap water, and stained with 0.4% (w/v) SRB in 1% acetic acid
(50 mL per well) for 30 min at room temperature. The cells were
then rinsed three times in 1% acetic acid to remove the unbound
Chemicals and drugs for in vitro assays in MDR cancer cells:
RPMI 1640 medium, antibiotic–antimycotic solution, l-glutamine,
and trypsin/EDTA were purchased from PAA (Vienna, Austria). Fetal
bovine serum (FBS), dimethyl sulfoxide (DMSO), and sulforhodami-
ne B (SRB) were obtained from Sigma–Aldrich Chemie GmbH (Ger-
many). Propidium iodide (PI) and annexin-V–FITC (AV) were pur-
chased from Abcam (Cambridge, UK). Doxorubicin (DOX) solution
was obtained from EBEWE Arzneimittel GmbH (Vienna, Austria), di-
luted in sterile water, and 1 mm aliquots were thawed from ꢁ208C
before use. GSK690693 was kindly provided by SelleckChem (Hous-
ton, TX, USA). GSK690693, as well as 9 and 10, were diluted in
DMSO, and 10 mm aliquots were stored at ꢁ208C.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Mori, Mattia
