BULLETIN OF THE
Article
Repositioning Irsogladine to Hsp90 Inhibitor
KOREAN CHEMICAL SOCIETY
triplicate wells. All experimental data were analyzed using
Prism GraphPad software.
of60_60_60pointsand0.375 Åspacingwaschosenforligand
docking experiments. The docking parameters consisted of
setting the population size to 150, the number of generations
to 27 000, and the number of evaluations to 25 000 000, while
the number of docking runs was set to 50 with a cutoff of 1 Å
for the root-mean-square tolerance for the grouping of each
docking run. The docking model of human Hsp90 with irso-
gladine is depicted in Figure 7, the rendering of which was
done using PyMol (DeLano Scientific).
Cell Culture. H1975 cells were grown in RPMI 1640 with L-
glutamine supplemented with streptomycin (500 mg/mL),
penicillin (100 units/mL), and 10% fetal bovine serum
(FBS). Cells were grown to confluence in a humidified atmos-
phere (37 ꢀC, 5% CO2).
Effect of Irsogladine on Cell Proliferation. Cells were
seeded at 3000 cells per well in a clear 96-well plate, the
medium volume was brought to 100 μL, and the cells were
allowed to attach overnight. The next day, varying concentra-
tions of irsogladine or 1% DMSO vehicle control were added
to the wells. Cells were then incubated at 37 ꢀC for 24 h. Cell
viability was determined using the Promega Cell Titer
96 Aqueous One Solution cell proliferation assay. After incu-
bation with the compounds, 20 μL of the assay substrate
solution was added to the wells, and the plate was incubated
at 37 ꢀC for an additional 1 h. Absorbance at 490 nm was then
read on the Tecan Infinite F200 Pro plate reader (Tecan,
Männedorf, Switzerland), and values were expressed as per-
centage absorbance from cells incubated in DMSO alone.
Western Blot. Cells were seeded in 60-mm culture dishes
(5 × 105/dish), and allowed to attach overnight. Irsogladine
was added at the concentrations indicated in Figure 6, and
the cells were incubated for an additional 48 h. For compari-
son, cells were also incubated with DMSO (1%) or geldana-
mycin (1 μM) for 24 h. Cells were harvested in ice-cold
lysis buffer (23 mM Tris–HCl pH 7.6, 130 mM NaCl, 1%
NP40, 1% sodium deoxycholate, 0.1% SDS), and 20 μg of
lysate per lane was separated by SDS-PAGE and followed
by transfer to a PVDF membrane (Bio-Rad, Hercules, CA,
USA). The membrane was blocked with 5% skim milk in
TBST, and then incubated with the corresponding antibody
(Her2, Akt, Cdk4, Hsp90, Hsp70, or β-Actin). After binding
of an appropriate secondary antibody coupled to horseradish
peroxidase, proteins were visualized by electrochemical lumi-
nescence (ECL) according to the instructions of the manufac-
turer (Thermo Scientific, Waltham, MA, USA).
Docking Studies. In silico docking of irsogladine with the 3D
coordinates of the X-ray crystal structures of the N-terminal
domain of human Hsp90 ((PDB code: 2XJX) was accom-
plished using the AutoDock program downloaded from the
Molecular Graphics Laboratory of the Scripps Research Insti-
tute. The AutoDock program was chosen because it uses a
genetic algorithm to generate the poses of the ligand inside
a known or predicted binding site utilizing the Lamarckian
version of the genetic algorithm, where the changes in confor-
mations adopted by molecules after in situ optimization are
used as subsequent poses for the offspring. In the docking
experiments carried out, water was removed from the 3D
X-ray coordinates, while Gasteiger charges were placed on
the X-ray structures of the N-terminal domain of Hsp90 along
with irsogladine using tools from the AutoDock suite. A grid
boxcenteredontheN-terminalHsp90domainwithdefinitions
SupportingInformation. Additionalsupportinginformation
is available in the online version of this article.
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Bull. Korean Chem. Soc. 2015, Vol. 36, 1495–1499
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